Novel adenoviruses, nucleic acids that code for the same and the use of said viruses

ABSTRACT

The present invention is related to an adenovirus expressing a first protein which is selected from the group comprising an E1B protein and an E4 protein, priorto a second protein which is selected from the group comprising an E1A protein.

The invention is related to adenoviruses, nucleic acids coding thereforand use thereof, in particular for the manufacture of a medicament forthe treatment of tumors.

A number of therapeutic concepts are currently used in the treatment oftumors. Apart from using surgery, chemotherapy and radiotherapy arepredominant. All these techniques are, however, associated withconsiderable side effects. The use of replication selective oncolyticviruses provides for a new platform for the treatment of tumors. Inconnection therewith a selective intratumor replication of a viral agentis initiated which results in virus replication, lysis of the infectedtumor cell and spreading of the virus to adjacent tumor cells. As thereplication capabilities of the virus is limited to tumor cells, normaltissue is spared from replication and thus from lysis by the virus.

For the time being, several viral systems are subject to clinic trialsaiming at tumor lysis. One example for such an adenovirus is dl1520(Onyx-015) which has been successfully used in clinical phases I and II(Khuri, F. et al. Nature Medicine 6, 879-885, 2000). Onyx-015 is anadenovirus having a completely deleted E1B-55 kDa gene. The completedeletion of the E1B55 kDa protein of the adenovirus is based on thediscovery that replication and thus lysis of cells is possible with anadenoviral vector which have a p53 deficiency (Kirn, D. et al., Proc.Am. Soc. Clin. Oncol. 17, 391a, 1998), whereby normal cells are notharmed. More particularly, the E1B-55 kDa gene product is involved inthe inhibition of p53, the transport of viral mRNA and the switching offof the protein synthesis of the host cell. The inhibition of p53 occursvia formation of a complex consisting of p53 and the adenoviral encodedE1B-55 kDa protein and/or a complex consisting of E1B-55 kDa and E4orf6.p53, coded by TP53, is the starting point for a complex regulatorymechanism (Zambetti, G. P. et al., FASEB J. 7, 855-865, 1993), whichresults, among others, in an efficient inhibition of the cellularreplication of viruses like adenovirus. The gene TP 53 is deleted ormutated in about 50% of all human tumors which results in the absenceof—desired—apoptosis due to chemotherapy or radiation therapy resultingin an usually unsuccessful tumor treatment.

A further concept of tumorlytic adenoviruses is based on the discoverythat if the E1A protein is present in a specific deleted form orcomprises one or several mutations, which do not affect the binding ofRb/E2F and/or p107/E2F and/or p130/E2F, such adenovirus will not inducethe entry of the infected cells into the S phase and will be capable ofreplicating in tumor cells which do not have a functional Rb protein.Additionally, the E1A protein can be deleted at the N-terminus andcomprise one or several mutations in the region of amino acid positions1 to 76 of the E1A proteins, respectively, in order to inhibit thebinding of E1A to p300 and thus to provide for a more selectivereplication in tumor cells. These approaches are described in anexemplary manner in European patent EP 0 931 830. Examples for suchviruses are AdΔ24, dl922-947, E1Ad/01/07 and CB016 (Howe, J. A. et al.,Molecular Therapy 2, 485-495, 2000; Fueyo, J. et al., Oncogene 19, 2-12,2000; Heise, C. et al., Nature Medicine 6, 11341139, 2001; Balague, C.et al., J. Virol. 75, 7602-7611, 2001). These adenoviral systems foroncolysis known in the prior art thus comprise distinct deletions in theE1A protein, whereby such deletions had been made under the assumptionthat a functional Rb protein and complexes consisting of inactive Rbprotein and E2F, respectively, would block an efficient in vivoreplication and in order to provide an adenoviral replication in vivo inRb-negative/mutated cells only. These adenoviral systems according tothe prior art are based on E1A in order to control in vivo replicationby means of the early E2 promoter (engl. E2 early promoter) and free E2F(Dyson, N. Genes & Development, 12, 2245-2262, 1998).

A further form of tumorlytic adenoviral systems is based on the use ofselective promoters for specifically expressing the viral oncogene E1Awhich provides for a selective replication in tumor cells (Rodriguez, R.et al., Cancer Res. 57, 2559-2563, 1997).

As described above, the selection of a cellular background which isappropriate for the respective concept underlying the mode of action isimportant for the various concepts of adenoviral tumorlytic viruses. Inother words, the various adenoviral systems currently known may only beused if distinct molecular biological prerequisites are realized. Thislimits the use of such systems to distinct patient groups.

A particular problem in the treatment of tumor diseases arises once thepatients develop a so-called multidrug resistance (engl. multidrugresistance (MDR)) which represents a particularly well studied form ofresistance of tumors against cytostatics (Gottesman and Pastan, Annu.Rev. Biochem. 62, 385-427, 1993). It is based on the overexpression ofthe membrane-bound transport protein P-glycoprotein which belongs to theso-called ABC transporters (Stein, U. et al., JBC 276, 28562-69, 2001,J. Wijnholds, Novartis Found Symp., 243, 69-79, 2002). Bargou, R. C. etal. and Oda, Y. et al (Bargou, R. C. et al., Nature Medicine 3, 447-450,1997; Clin. Cancer Res. 4, 2273-2277, 1998) were able to show thatnuclear localisation of the human transcription factor YB-1 is directlyinvolved in the activation of the expression of the P-glycoprotein.Further studies confirmed that YB-1 is transported into the nucleus byvarious stress conditions such as UV irradiation, administration ofcytostatics (Koike, K. et al., FEBS Lett 17, 390-394, 1997) andhyperthermia (Stein, U. et al., JBC 276, 28562-69, 2001). Furtherstudies confirmed that the nuclear localisation of YB-1 has an impact onone further ABC transporter. This ABC transporter is referred to as MRP(engl. multidrug resistance-related protein) and is involved in theformation of the so-called atypical non-P-glycoprotein dependentmultidrug resistance (Stein, U. et al., JBC 276, 28562-69, 2001).

The problem underlying the present invention is to provide a technicalteaching and in particular a means which allows to treat an organism,more particularly a human organism and a group of patients,respectively, specifically with tumorlytically active agents. It is afurther problem underlying the present invention to provide a meanswhich is suitable to cause tumorlysis in patients having tumor diseaseswhich are resistant to cytostatics, particularly those which have amultidrug resistance. Finally, a problem underlying the presentinvention is to provide for an adenovirus which is suitable for celllysis.

In a first aspect the problem underlying the invention is solved by anadenovirus expressing a first protein which is selected from the groupcomprising an E1B protein and an E4 protein, prior to a second proteinwhich is selected from the group comprising an E1A-protein.

In an embodiment the first protein is an E1B protein, preferably anE1B55 kd protein.

In a further embodiment the first protein is an E4 protein, preferablyan E4orf6 protein.

In a preferred embodiment the first protein is a combination of E1Bprotein and E4 protein, preferably a combination of E1B55 kD protein andE4orf6 protein.

In a preferred embodiment the E1A protein is an E1A12S protein. In analternative embodiment the E1A protein is the E1A protein of thewildtype adenovirus, preferably of Ad 5, or the E1A of adenovirus delta24.

In a second aspect the problem underlying the invention is solved by anadenovirus, whereby the adenovirus comprises at least one nucleic acidcoding for a protein which is selected from the group comprising E1Bproteins, E4 proteins and E1A proteins, whereby the at least one proteinis under the control of a promoter which is different from the promotercontrolling the expression of the protein in a wildtype adenovirus.

In an embodiment of the second aspect the adenovirus is an adenovirusaccording to the first aspect of the present invention.

In an embodiment of the second aspect the at least one protein is an E1Bprotein, preferably an E1B55 kD protein.

In an embodiment of the second aspect the at least one protein is an E4protein, preferably an E4orf6 protein.

In an embodiment of the second aspect the at least one protein is an E1Aprotein, preferably an E1A12S protein. In a particularly preferredembodiment the E1A12S protein is a E1A12S protein of Ad5, preferably ofwildtype Ad5, or a E1A12S protein of adenovirus delta 24.

In an embodiment of the second aspect the at least one protein is acombination of E1B protein and E4 protein, preferably a combination ofE1B55 kD protein and E4orf6 protein.

In an embodiment of the second aspect the at least one protein is acombination of E1B protein and E1A protein, preferably a combination ofE1B55 kD protein and E1A12S protein.

In a preferred embodiment of the second aspect the at least one proteinis a combination of E4 protein and E1A protein, preferably a combinationof E4orf6 protein and E1A12S protein.

In an embodiment of the second aspect the at least one protein is acombination of E1B protein, E4 protein and E1A protein, preferably acombination of E1B55 kD protein, E4orf6 protein and E1A12S protein.

In an embodiment of the second aspect the expression of the E1B proteinis controlled by a promoter, whereby the promoter is selected from thegroup comprising tumor-specific promoters, organ-specific promoters,tissue-specific promoters, heterologous promoters and adenoviralpromoters, whereby the adenoviral promoter is different from the E1Bpromoter.

In an embodiment of the second aspect the expression of the E4 proteinis controlled by a promoter, whereby the promoter is selected from thegroup comprising tumor-specific promoters, organ-specific promoters,tissue-specific promoters, heterologous promoters and adenoviralpromoters, whereby the adenoviral promoter is different from the E4promoter.

In a preferred embodiment of the second aspect the adenoviral promoteris the E1A promoter.

In an embodiment of the second aspect the expression of the E1A proteinis controlled by a promoter, whereby the promoter is selected from thegroup comprising tumor-specific promoters, organ-specific promoters,tissue-specific promoters, heterologous promoters and adenoviralpromoters, whereby the adenoviral promoter is different from the E1Apromoter.

In a preferred embodiment of the second aspect the promoter controllingthe expression of the E1A protein is YB-1 controlled or can be regulatedby YB-1.

In a preferred embodiment of the second aspect the promoter controllingthe expression of the E1A protein is the adenoviral E2 late promoter.

In an embodiment of the first and second aspect the E4 protein,preferably the E4orf6 protein, and the E1B protein, preferably the E1B55kd protein, are under the control of the same or a mutual promoter.

In a third aspect the problem underlying the invention is solved by anadenovirus, whereby the adenovirus provides YB-1 in the nucleus throughat least one adenoviral protein or that the provision of YB-1 in thenucleus is mediated through at least one adenoviral protein, wherebypreferably the adenoviral protein is different from E1A.

In an embodiment of the third aspect, the adenovirus is an adenovirusaccording to the first and/or second aspect of the present invention.

In a fourth aspect the problem underlying the invention is solved by anadenovirus, whereby the adenovirus provides YB-1 for adenoviralreplication through at least one adenoviral protein or mediates theprovision of YB-1 for adenoviral replication through at least oneadenoviral protein, whereby preferably the adenoviral protein isdifferent from E1A.

In an embodiment of the fourth aspect, the adenovirus us an adenovirusaccording to the first and/or second and/or third aspect of the presentinvention.

In an embodiment of the third and the fourth aspect, the adenoviralprotein is a complex of E4orf6 and E1B55 kd.

In a fifth aspect the problem underlying the invention is solved by anadenovirus, whereby the nucleic acid of the adenovirus comprises atleast one functionally inactive adenoviral region, whereby the region isselected from the group comprising the E1 region, the E3 region, the E4region and combinations thereof.

In an embodiment of the fifth aspect the adenovirus is an adenovirus inaccordance with the first and/or second and/or third and/or fourthaspect of the present invention.

In an embodiment of the fifith aspect the region is the E1 region.

In an embodiment of the fifth aspect the region is the E3 region.

In an embodiment of the fifth aspect the region is the E4 region.

In an embodiment of the fifth aspect the region comprises the E1 region,the E3 region and the E4 region.

In a sixth aspect the problem underlying the invention is solved by anadenovirus, whereby the adenovirus comprises at least one expressioncassette, whereby the expression cassette comprises at least onepromoter and a nucleic acid coding for an adenoviral protein, wherebythe adenoviral protein is an E1B protein, preferably an E1B55 kDprotein.

In an embodiment of the sixth aspect the adenovirus is an adenovirusaccording to the first and/or second and/or third and/or fourth and/orfifth aspect of the present invention.

In an embodiment of the sixth aspect the promoter is different from theE1B promoter.

In an embodiment of the sixth aspect the promoter is selected from thegroup comprising tumor-specific promoters, organ-specific promoters,tissue-specific promoters, heterologous promoters and adenoviralpromoters, whereby the promoter is different from the E1B promoter.

In a seventh aspect the problem underlying the invention is solved by anadenovirus, whereby the adenovirus comprises at least one expressioncassette, whereby the expression cassette comprises at least onepromoter and a nucleic acid coding for an adenoviral protein, wherebythe adenoviral protein is an E4 protein, preferably an E4orf6 protein.

In an embodiment of the seventh aspect the adenovirus is an adenovirusaccording to the first and/or second and/or third and/or fourth and/orfifth and/or sixth aspect of the present invention.

In an embodiment of the seventh aspect the promoter is selected from thegroup comprising tumor-specific promoters, organ-specific promoters,tissue-specific promoters, heterologous promoters and adenoviralpromoters, whereby the adenoviral promoters are different from the E4promoter.

In an embodiment of the seventh aspect the promoter is the E1A promoter.

In an eighth aspect the problem underlying the invention is solved by anadenovirus, whereby the adenovirus comprises at least one expressioncassette, whereby the expression cassette comprises at least onepromoter and a nucleic acid coding for an adenoviral protein, wherebythe adenoviral protein is an E1A protein, preferably an E1A12S protein.

In an embodiment of the eighth aspect, the adenovirus is an adenovirusaccording to the first and/or second and/or third and/or fourth and/orfifth and/or sixth and/or seventh aspect of the present invention.

In an embodiment of the eighth aspect the promoter is different from theE1A promoter.

In an embodiment of the eighth aspect the promoter is selected from thegroup comprising tumor-specific promoters, organ-specific promoters,tissue-specific promoters, heterologous promoters and adenoviralpromoters.

In an embodiment of the first and/or second and/or third and/or fourthand/or fifth and/or sixth and/or seventh and/or eighth aspect theadenovirus comprises a nucleic acid, whereby the nucleic acid codes forYB-1.

In a preferred embodiment of the eighth aspect the nucleic acid codingfor YB-1 is under the control of a promoter, whereby the promoter ispreferably the E2 late promoter.

In an embodiment of the eighth aspect the nucleic acid coding for YB-1is under the control of a promoter, whereby the promoter is YB-1dependent and YB-1 controlled, respectively.

In an embodiment of the eighth aspect the nucleic acid coding for YB-1is part of the expression cassette comprising a nucleic acid coding foran E1A protein, preferably a nucleic acid coding for an E1A12S protein.

In an embodiment of the eighth aspect the nucleic acid coding for theE1A protein is separated from the nucleic acid coding for YB-1 throughan IRES sequence.

In an embodiment of the sixth and/or seventh and/or eighth aspect thenucleic acid coding for the E4 protein, preferably the E4orf6 protein,and the nucleic acid coding for the E1B protein, preferably the E1B55 kDprotein, are contained in an expression cassette, whereby preferably thetwo coding sequences are separated through an IRES sequence.

In a preferred embodiment of the eighth aspect the promoter of theexpression cassette is selected from the group comprising tumor-specificpromoters, organ-specific promoters, tissue-specific promoters,heterologous promoters and adenoviral promoters, whereby the adenoviralpromoters are different from the E4 promoter and different from the E1Bpromoter, preferably different from the wildtype E4 promoter anddifferent from the wildtype E1B promoter.

In an embodiment of the first and/or second and/or third and/or fourthand/or fifth and/or sixth and/or seventh and/or eighth aspect theadenovirus comprises an expression cassette comprising a promoter and anucleic acid sequence, whereby the nucleic acid sequence is selectedfrom the group comprising aptamers, ribozymes, aptazymes, antisensemolecules and siRNA.

In an embodiment of the first and/or second and/or third and/or fourthand/or fifth and/or sixth and/or seventh and/or eighth aspect theadenovirus comprises an expression cassette comprising a promoter and anucleic acid sequence, whereby the nucleic acid sequence is a codingnucleic acid, whereby the nucleic acid codes for a molecule which isselected from the group comprising peptides, polypeptides, proteins,anticalines, antibodies and antibody fragments.

In an embodiment of the first and/or second and/or third and/or fourthand/or fifth and/or sixth and/or seventh and/or eighth aspect theadenovirus comprises an expression cassette, whereby the expressioncassette comprises a promoter and a nucleic acid sequence, whereby thenucleic acid sequence is selected from the group comprising apoptosisinducing genes, prodrug genes, protease inhibitors, tumor suppressorgenes, cytokines and angiogenesis inhibitors.

In an embodiment of the first and/or second and/or third and/or fourthand/or fifth and/or sixth and/or seventh and/or eighth aspect theadenovirus is a recombinant adenovirus.

In an embodiment of the first and/or second and/or third and/or fourthand/or fifth and/or sixth and/or seventh and/or eighth aspect theadenovirus is an adenovirus mutant.

In an embodiment of the first and/or second and/or third and/or fourthand/or fifth and/or sixth and/or seventh and/or eighth aspect theadenovirus is replication deficient.

In an embodiment of the first and/or second and/or third and/or fourthand/or fifth and/or sixth and/or seventh and/or eighth aspect theadenovirus is capable of replicating in cells comprising deregulatedYB-1 or having YB-1 in the nucleus.

In an embodiment of the first and/or second and/or third and/or fourthand/or fifth and/or sixth and/or seventh and/or eighth aspect the cellscontain YB-1 in the nucleus independent of the cell cycle.

In an embodiment of the first and/or second and/or third and/or fourthand/or fifth and/or sixth and/or seventh and/or eighth aspect theadenovirus does not comprise any E1A13S protein and/or the adenovirusdoes not comprise any nucleic acid coding for a E1A13S protein.

In a ninth aspect the problem underlying the invention is solved by anucleic acid coding for an adenovirus according to any of the aspectsone to eight.

In a tenth aspect the problem underlying the invention is solved byreplication system comprising a nucleic acid according to the ninthaspect and a nucleic acid of a helper virus, whereby the nucleic acid ofthe helper virus comprises one or more of the expression cassettes ofthe adenovirus according to any of the aspects one to eight.

In an embodiment of the tenth aspect the adenovirus or the nucleic acidcoding therefor is lacking the expression cassette comprised by thehelper virus.

In an eleventh aspect the problem underlying the invention is solved bya vector comprising a nucleic acid according to the ninth aspect and/ora replication system according to the tenth aspect.

In an embodiment of the eleventh aspect the vector is an expressionvector.

In a twelfth aspect the problem underlying the invention is solved by anadenovirus ell comprising an adenovirus according to any of aspects oneto eight and/or a nucleic acid according to the ninth aspect and/or areplication system according to the tenth aspect and/or a vectoraccording to the eleventh aspect.

In an embodiment of the twelfth aspect the cell is a eucaryotic cell,preferably an animal cell, more preferably a mammalian cell.

In a preferred embodiment of the twelfth aspect the mammalian cell is acell selected from the group comprising cells of mice, rats, guineapigs, pigs, sheep, goats, cattle, horses, dogs, cats and human beings.

In a thirteenth aspect the problem underlying the invention is solved byan organism, preferably a mammal organism, comprising an adenovirusaccording to aspect one to eighth, a nucleic acid according to the ninthaspect, a replication system according to the tenth aspect, a vectoraccording to the eighth aspect or a cell according to the twelfthaspect, whereby the organism is preferably selected from the groupcomprising mice, rats, guinea pigs, pigs, sheep, goats, cattle, horses,dogs and cats.

In a fourteenth aspect the problem underlying the invention is solved bythe use of an adenovirus according to any of the aspects one to eighth,a nucleic acid according to the ninth aspect, a replication systemaccording to the tenth aspect, a vector according to the eighth aspect,or a cell according to the twelfth aspect, for replication of anadenovirus, preferably for in vitro replication of an adenovirus.

In a fifteenth aspect the problem underlying the invention is solved bythe use of an adenovirus according to any of aspects one to eighth, anucleic acid according to the ninth aspect, a replication systemaccording to the tenth aspect, a vector according to the eight aspect,or a cell according to the twelfth aspect for the manufacture of anadenovirus, preferably for in vitro manufacture of an adenovirus.

In a sixteenth aspect the problem underlying the invention is solved bythe use of an adenovirus according to any of aspects one to eight, anucleic acid according to the ninth aspect, a replication systemaccording to the tenth aspect, a vector according to the eighth aspect,or a cell according to any the twelfth aspect for the expression ofgenes, preferably of genes which promote cell lysis, preferably celllysis during adenoviral replication, and/or are promoting adenoviralmediated cell lysis.

In an embodiment of the sixteenth aspect the expressed genes aretransgenes as disclosed herein.

In a seventeenth aspect the problem underlying the invention is solvedby the use of an adenovirus according to any of aspects one to eight, anucleic acid according to the ninth aspect, a replication systemaccording to the tenth aspect, a vector according to the eighth aspect,or a cell according to the twelfth aspect for the manufacture of amedicament.

In an embodiment of the fourteenth to the seventeenth aspect the cell inwhich the adenovirus replicates, has YB-1 in its nucleus, preferably hasYB-1 in its nucleus independent of the cell cycle.

In an embodiment of the fourteenth to the seventeenth aspect the cell inwhich the adenovirus replicates, comprises deregulated YB-1.

In an embodiment of the use of the seventeenth aspect the medicament isfor the treatment of tumor diseases.

In a preferred embodiment of the use of the seventeenth aspect the tumordisease is selected from the group comprising malignant diseases,cancer, cancer diseases and tumors.

In an embodiment of the use of the seventeenth aspect the tumors areselected from the group comprising solids, non-solid, malignant andbenign tumors.

In an embodiment of the use of the seventeenth aspect at least a part ofthe tumor forming cells have YB-1 in the nucleus, preferably have YB-1in the nucleus independent of the cell cycle.

In an embodiment of the use of the seventeenth aspect at least a part ofthe cells forming the tumor comprises deregulated YB-1.

In an embodiment of the use of the seventeenth aspect at least a part ofthe cells forming the tumor are Rb positive or Rb negative.

In an embodiment of the use of the seventeenth aspect at least a part ofthe cells forming the tumor have a resistance, preferably a multipleresistance against pharmaceutically active agents.

In a preferred embodiment of the use of the seventeenth aspect theresistance is a multiple resistance.

In an embodiment of the use of the seventeenth aspect the resistance isagainst anti-tumor agents, preferably cytostatics, and/or that theresistance is caused by irradiation.

In an embodiment of the use of the seventeenth aspect the patient forwhich the medicament is intended, comprises a plurality of cells,whereby the cells are cells as described in the various embodiments ofthe use according to the seventeenth aspect of the present invention.

In an embodiment of the use of the seventeenth aspect the medicamentcomprises at least one further pharmaceutically active agent.

In an embodiment of the use of the seventeenth aspect the medicament isadministered together with a further pharmaceutically active agent or isintended therefor.

In an embodiment of the use of the seventeenth aspect the furtherpharmaceutically active agent is selected from the group comprisingcytokines, metalloproteinase inhibitors, angiogenesis inhibitors,cytostatics such as Irinotecan and CPT-11 against colorectal carcinomaand Daunorubicin against leukemia, cell cycle inhibitors such as CYC202which inhibits CDK2/CyclinE kinase activity and can be used againstcolorectal tumors (McClue S J, Int. J. Cancer 2002, 102, 463-468) andBAY 43-9006 which inhibits Raf-1 and is, for example, effective againstmamma carcinoma (Wilhelm S M et al., Cancer Res. 2004, 64, 7099-7109),proteosome inhibitors such as PS-341 which inhibits the 26S proteasomeactivity and is used against squamous-cell carcinoma (Fribley A et al.,Mol Cell Biol 2004 November; 24(22): 9695-704), recombinant antibodiessuch as against the EGF receptor (Herceptin for breast carcinoma andprostate tumor; H. G. van der Poel, European Urology 2004, 1-17; Erbituxagainst head and neck tumors; Bauman M et al., Radiother. Oncol., 2004,72, 257-266), and inhibitors of the signal transduction cascade such asSTI 571 which represses, among others, c-kit and can be used againstgastrointestinal tumors (H. G. van der Poel, European Urology 2004, 45,1-17), ABT-627, an endothelin inhibitor, which may be used, amongothers, against prostate tumors (H. G. van der Poel, European Urology2004, 45, 1-17), SU5416 which inhibits phosphorylation of the VEGFtyrosine kinase receptor and which may be used, among others, againstglioblastoma and prostate cancer (Bischof M et al Int. J. Radiat. Oncol.Biol. Phys. 2004; 60 (4): 1220-32), ZD1839 which inhibits EGFR tyrosineactivity and may be used, among others, against prostate tumors (H. G.van der Poel, European Urology 2004, 45, 1-17); rapamycin derivativessuch as CCI-779 and RAD001 which inhibit mTOR and can be used againstprostate tumors. It is within the present invention that the variousadenoviruses described herein and the adenoviruses to be used inaccordance with the present invention, respectively, can, in principle,be used with each and any of the aforementioned compounds for each andany of the indication described herein in connection therewith. In aparticularly preferred embodiment the indication is the one which isdescribed for any of the previously mentioned pharmaceutically activecompounds.

In an embodiment of the use of the seventeenth aspect the medicament isadministered prior, during or after irradiation.

In a preferred embodiment of the use of the seventeenth aspect theradiation is administered for the purpose of treating a tumor.

In an embodiment of the use of the seventeenth aspect the cell or theorganism to be treated is subject to a measure, whereby the measure isselected from the group comprising irradiation, administration ofcytostatics and hyperthermia.

In an embodiment of the use of the seventeenth aspect the measure isapplied locally or systemically.

In an embodiment of the use of the seventeenth aspect the irradiationuses high-energy radiation, preferably uses any irradiation as used inthe treatment of tumor diseases.

In an eighteenth aspect the problem underlying the invention is solvedby the use of an adenovirus according to any of the aspects one toeight, a nucleic acid according to the ninth aspect, a replicationsystem according to the tenth aspect, a vector according to the eleventhaspect, or a cell according to the twelfth aspect for the manufacture ofa medicament for the treatment of tumor diseases, characterised in thatthe tumor disease is selected from the group comprising breast tumors,bone tumors, gastric tumors, intestinal tumors, gall-bladder tumors,pancreas tumors, liver tumors, kidney tumors, brain tumors, ovariantumors, skin tumors, tumors of cutaneous appendages, head and neckcancer, uterine tumors, synovial tumors, laryngeal tumors, oesophagealtumors, lingual tumors, prostate tumors, preferably one of the precedingtumor diseases having the characteristics as described in any of thepreceding claims.

In a nineteenth aspect the problem underlying the invention is solved bythe use of an adenovirus according to any of the aspects one to eight, anucleic acid according to the ninth aspect, a replication systemaccording to the tenth aspect, a vector according to the eleventhaspect, or a cell according to the twelfth aspect for the manufacture ofmedicament for the treatment of tumor diseases, whereby thetumor-specific promoter is a promoter which is specific for the tumorfor which the medicament is used.

In a twentieth aspect the problem underlying the invention is solved bya pharmaceutical composition comprising an adenovirus according to anyof the aspects one to eight, a nucleic acid according to the ninthaspect, a replication system according to the tenth aspect, a vectoraccording to the eleventh aspect, or a cell according to the twelfthaspect and optionally a pharmaceutically acceptable carrier.

In a twenty-first aspect the problem underlying the present invention issolved by the use of a virus, preferably an adenovirus, for themanufacture of a medicament, whereby the virus is replication deficientin normal cells which do not contain YB-1 in the nucleus, in cells whichdo not contain YB-1 in the nucleus independent of the cell cycle, and incells which do not contain deregulated YB-1, respectively, and the viruscodes for an oncogene or oncogene product, in particular an oncogeneprotein which at least transactivates one viral gene in YB-1 nucleuspositive cells, preferably an adenoviral gene, whereby the gene isselected from the group comprising E1B55 kDa, E4orf6, E4 orf3 and E3ADP.Preferably, the virus expresses the viral proteins E1B55 kD, which isalso referred to herein as E1B55 kDa, and E4orf6.

In a twenty-second aspect the problem underlying the invention is solvedby the use of a virus, preferably an adenovirus, for replication incells, which contain YB-1 in the nucleus, whereby the virus isreplication deficient in cells which do not contain YB-1 in the nucleus,or cells which do not contain YB-1 in the nucleus independent of thecell cycle, or cells which do not contain any deregulated YB-1, andwhereby the virus codes for an oncogene or an oncogene product, inparticular an oncogene protein, which transactivates at least one viralgene, preferably an adenoviral gene, whereby the gene is selected fromthe group comprising E1B55 kDa, E4orf6, E4orf3 and E3ADP.

In an embodiment of the uses in accordance with the twenty-first andtwenty-second aspect of the invention the virus, in particular theadenovirus, replicates in cells which contain YB-1 in the nucleus orwhich do not contain YB-1 in the nucleus independent of the cell cycle,or which do not comprise any deregulated YB-1.

In a further embodiment of the uses in accordance with the twenty-firstand twenty-second aspect of the invention the viral oncogene protein isE1A and/or the oncogene is the gene coding for E1A and/or the oncogeneprotein is E1A.

In a preferred embodiment the viral oncogene protein E1A is capable ofbinding a functional Rb tumor suppressor gene product.

In an alternative embodiment the viral oncogene protein E1A is incapableof binding a functional Rb tumor suppressor gene product.

In a further embodiment of the uses in accordance with the twenty-firstand twenty-second aspect of the invention the viral oncogene protein E1Adoes not induce nuclear localisation of YB-1.

In a further embodiment of the uses in accordance with the twenty-firstand twenty-second aspect of the invention the medicament is for patientsthe cells of which are either Rb positive or Rb negative.

In a preferred embodiment the cells are those cells involved in theformation of the condition which is to be affected by the medicament.

In a further embodiment of the uses in accordance with the twenty-firstand twenty-second aspect of the invention the cells are Rb-negative andYB-1-positive in the nucleus, in particular are YB-1 positive in thenucleus independent of the cell cycle.

In a still further embodiment of the uses in accordance with thetwenty-first and twenty-second aspect of the invention the medicament isfor the treatment of tumors.

In a still further embodiment of the uses in accordance with thetwenty-first and twenty-second aspect of the invention the cells, inparticular the cells forming the tumor or parts thereof, are resistant,in particular multiple resistant against drugs, preferably anti-tumoragents and more preferably cytostatics.

In a preferred embodiment of the uses in accordance with thetwenty-first and twenty-second aspect of the invention the cells areexpressing, preferably are over-expressing the membrane-bound transportprotein P-glycoprotein and/or MRP.

In a further embodiment of the uses in accordance with the twenty-firstand twenty-second aspect of the invention the cells are eitherp53-positive or p53-negative.

In an embodiment of the uses in accordance with the twenty-first andtwenty-second aspect of the invention the oncogene protein comprises,compared to the wildtype oncogene protein E1A, one or several mutationsor deletions, whereby the deletions are preferably those selected fromthe group comprising deletions of the CR3 region and deletions of theN-terminus and deletions of the C-terminus. It is contemplated that theE1A oncogene protein is capable of binding to Rb.

In a further embodiment of the uses in accordance with the twenty-firstand twenty-second aspect of the invention the oncogene proteincomprises, compared to the wildtype oncogene protein, one or severalmutations or deletions, whereby the deletion is preferably one in theCR1 region and/or CR2 region. It is contemplated that the oncogeneprotein E1A is incapable of binding to Rb.

In an embodiment of the uses in accordance with the twenty-first andtwenty-second aspect of the invention the viral oncogene protein, inparticular E1A, is under the control of a tissue-specific and/ortumor-specific promoter.

In a further embodiment of the uses in accordance with the twenty-firstand twenty-second aspect of the invention the virus, in particular theadenovirus, is coding for YB-1.

In a still further embodiment of the uses in accordance with thetwenty-first and twenty-second aspect of the invention YB-1 is under thecontrol of a tissue-specific and/or tumor-specific promoter.

In a preferred embodiment of the uses in accordance with thetwenty-first and twenty-second aspect of the invention the virus, inparticular the adenovirus, codes for at least one protein which isselected from the group comprising E4orf6, E4 orf3, E1B55k andadenoviral E3ADP protein.

In a alternative embodiment of the uses in accordance with thetwenty-first and twenty-second aspect of the invention the cells containYB-1 in the nucleus, in particular the cells forming the tumor or partthereof comprise YB-1 in the nucleus.

In a further embodiment of the uses in accordance with the twenty-firstand twenty-second aspect of the invention the tumor contains YB-1 in thenucleus after induction of the transport of YB-1 into the nucleus.

In a preferred embodiment of the uses in accordance with thetwenty-first and twenty-second aspect of the invention the transport ofYB-1 into the nucleus is caused by at least one measure, whereby themeasure is selected from the group comprising irradiation,administration of cytostatics and hyperthermia.

In a particularly preferred embodiment of the uses in accordance withthe twenty-first and twenty-second aspect of the invention the measureis applied to a cell, an organ or an organism.

In a preferred embodiment of the uses in accordance with thetwenty-first and twenty-second aspect of the invention the virus, inparticular the adenovirus, is selected from the group comprising AdΔ24,dl922-947, E1Ad/01/07, dl1119/1131, CB 016, dl520 and viruses which arelacking an expressed viral E1A oncogene which is capable of binding afunctional Rb tumor suppressor gene product.

In a twenty-third aspect the problem is solved by the use of a virus,preferably the adenovirus, for the manufacture of a medicament, wherebythe virus, in particular the adenovirus, is adapted such that thereplication is controlled through or by YB-1 mediated activation of theE2-late promoter, preferably predominantly controlled by the activationof the E2-late promoter. In an embodiment YB-1 is either a transgenicYB-1 or a cellular YB-1, in particular a cellular deregulated YB-1 orderegulated YB-1. A transgenic YB-1 is preferably a YB-1 which isexpressed in a cell by a vector, in particular by a or the adenovirus.The E2-late promoter is preferably the adenoviral E2-late promoter ascontained in wildtype adenovirus, or an E2-late promoter as used inconnection with the expression of the transgens as described herein.

In a twenty-fourth aspect the problem is solved by the use of a virus,in particular the adenovirus, for replication in cells which containYB-1 in the nucleus, whereby the virus, in particular the adenovirus, isadapted such that the replication is controlled by YB-1 through theactivation of the E2-late promoter, preferably predominantly by theactivation of the E2-late promoter. In an embodiment the YB-1 is eithera transgenic YB-1 or a cellular YB-1, in particular a cellularderegulated or deregulated YB-1. A transgenic YB-1 is preferably a YB-1which is expressed in a cell by a vector, in particular a or theadenovirus. The E2-late promoter is preferably the adenoviral E2-latepromoter as present in wildtype adenovirus, or an E2-late promoter asused in connection with the expression of transgenes as describedherein.

In a preferred embodiment of the twenty-third and/or twenty-fourthaspect of the present invention the adenovirus is adapted as disclosedherein, particularly adapted such that it may be used in accordance withthe present invention.

In a twenty-fifth aspect the problem is solved by a viral oncogeneprotein, in particular an isolated viral oncogene protein, whereby theviral oncogene protein has the following characteristics:

-   -   a) transactivation of at least one viral gene in YB-1 nucleus        positive cells which is selected from the group comprising        E1B55k, E3ADP and E4orf6 and E4orf4; and    -   b) no induction of YB-1 in a cell nucleus, in particular in the        cell nucleus of the cell in which the viral oncogene protein is        present.

In an embodiment the viral oncogene protein is E1A.

In a further embodiment the viral oncogene protein comprises, comparedto the wildtype oncogene protein, one or several mutations or deletions,whereby the deletions are preferably those selected from the groupcomprising deletion of the CR3 region, deletion of the N-terminus anddeletion of the C-terminus.

In an embodiment the induction of YB-1 through the viral oncogeneprotein does not occur under the proviso that E4orf6 and/or E1B55 kDis/are not present in the cell comprising said nucleus.

It is contemplated that the viral oncogene protein is capable of bindingto Rb.

In an alternative embodiment the viral oncogene protein comprises one orseveral mutations or deletions, whereby the deletion is preferably onein the CR1 region and/or the CR2 region of the E1A oncogene protein. Itis contemplated that the viral oncogene protein is incapable of bindingto Rb.

In a twenty-sixth aspect the invention is related to the use of a viralreplication system, in particular an adenoviral replication systemcomprising a nucleic acid coding for a virus, in particular anadenovirus, as used in accordance with the present invention, andcomprising a nucleic acid of a helper virus, whereby the nucleic acid ofthe helper virus comprises a nucleic acid sequence coding for YB-1.

In an embodiment the viral nucleic acid, in particular the adenoviralnucleic acid, and/or the nucleic acid of the helper virus is present asreplicable vector.

In a twenty-seventh aspect the invention is related to the use of anucleic acid coding for a virus, in particular an adenovirus, as used inaccordance with the present invention, for the manufacture of amedicament, in particular for the manufacture of a medicament for thetreatment of tumors.

In a preferred embodiment the cells, in particular the cells forming thetumor or parts thereof, show a resistance, in particular a multipleresistance against drugs, in particular anti-tumor agents and moreparticularly cytostatics.

In a twenty-eighth aspect the present invention is related to the use ofa nucleic acid coding for a virus, in particular an adenovirus, as usedin accordance with the present invention, for the replication in cellswhich contain YB-1 in the nucleus, whereby the virus is replicationdeficient in cells which do not contain YB-1 in the nucleus or which donot comprise YB-1 in the nucleus independent of the cell cycle or whichdo not comprise any deregulated YB-1, and whereby the virus codes for anoncogene or an oncogene protein which at least transactivates one viralgene, preferably an adenoviral gene in YB-1 nucleus positive cells,whereby the gene is selected from the group comprising E1B55 kDa,E4orf6, E4orf3 and E3ADP.

In a twenty-ninth aspect the problem is solved by the use of a nucleicacid coding for a virus, in particular an adenovirus, as used inaccordance with the invention, for the manufacture of a medicament,whereby the virus is adapted such that the replication is controlled byYB-1 through the activation of the E2-late promoter, preferablypredominantly through the activation of the E2-late promoter. In anembodiment the YB-1 is either a transgenic YB-1 or a cellular, inparticular cellular deregulated, or deregulated YB-1. A transgenic YB-1is preferably a YB-1 which is expressed in a cell by a vector, inparticular by a or the adenovirus. The E2-late promoter is preferablythe adenoviral E2-late promoter such as contained in wildtypeadenovirus, or an E2-late promoter as used in connection with theexpression of transgenes described herein.

In a thirtieth aspect the problem is solved by the use of a nucleic acidcoding for a virus, in particular an adenovirus, as used in accordancewith the present invention, for the replication in cells, whereby thevirus is adapted so that the replication is controlled by YB-1 throughthe activation of E2-late promoter, preferably predominantly through theactivation of the E2-late promoter. In an embodiment YB-1 is either atransgenic YB-1 or a cellular, in particular cellular deregulated YB-1.A transgenic YB-1 is preferably one which is expressed in a cell by avector, preferably by a or the adenovirus. The E2-late promoter ispreferably the adenoviral E2-late promoter as present in wildtypeadenovirus, or an E2-late promoter as used in connection with theexpression of transgenes as described herein.

In a thirty-first aspect the problem is solved by the use of a vectorcomprising one of the previously described nucleic acids, for the use inaccordance with the twenty-first or twenty-second aspect of the presentinvention.

In a thirty-second aspect the invention is related to the use of anagent interacting with YB-1 for the characterisation of cells, of cellsof a tumor tissue or of patients in order to determine whether theyshall be contacted and/or treated with a virus, in particular anadenovirus, as used in accordance with the present invention.

In an embodiment the agent is selected from the group comprisingantibodies, anticalines, aptamers, aptazymes and spiegelmers.

In a thirty-second aspect the problem is solved by the use of the viraloncogene protein in accordance with the present invention, or a nucleicacid coding therefor, for the manufacture of a virus, in particular ofan adenovirus, as used in connection with the uses in accordance withthe twenty-first and twenty-second aspect of the present invention.

In an embodiment the virus comprises a nucleic acid coding for atransgene.

In a further embodiment the virus comprises the translation productand/or the transcription product of a transgene.

In a preferred embodiment the nucleic acid of the adenoviral replicationsystem and/or the nucleic acid of the helper virus comprises a transgeneor a nucleic acid coding for a transgene.

In a still further embodiment the nucleic acid comprises a transgen or anucleic acid coding for a transgene.

In an alternative embodiment the transgene is selected from the groupcomprising prodrugs, cytokines, apoptosis-inducing genes, tumorsuppressor genes, genes for metalloproteinase inhibitors and genes forangiogenesis inhibitors and for tyrosine kinase inhibitors.

In an embodiment the transgene is selected from the group comprisingnucleic acids for siRNA, for aptamers, for antisense molecules and forribozymes, whereby the siRNA, the aptamers, the antisense moleculesand/or the ribozymes are directed against the target molecule.

In a further embodiment the target molecule is selected from the groupcomprising resistance relevant factors, anti-apoptosis factors,oncogenes, angiogenesis factors, DNA synthesis enzymes, DNA repairenzymes, growth factors and their receptors, transcription factors,metalloproteinases, in particular matrix metalloproteinases, andplasminogen activator of the urokinase type. In an embodiment theresistance relevant factors are preferably selected from the groupcomprising P-glycoprotein, MRP and GST, and also comprise the nucleicacids coding therefor. In an embodiment the anti-apoptosis factors areselected from the group comprising BCL2, and also comprise the nucleicacids coding therefor. In an embodiment the oncogenes are selected fromthe group comprising Ras, in particular mutated Ras, Rb and MYC, andalso comprise the nucleic acids coding therefor. In an embodiment theangiogenesis factors are selected from the group comprising VEGF and HMGproteins, and also comprise the nucleic acids coding therefor. In anembodiment the DNA synthesis enzymes are selected from the groupcomprising telomerase, and also comprise the nucleic acids codingtherefor. In an embodiment the DNA repair enzymes are selected from thegroup comprising Ku-80, and also comprise the nucleic acids codingtherefor. In an embodiment the growth factors are selected from thegroup comprising PDGF, EGF and M-CSF, and also comprise the nucleicacids coding therefor. In a further embodiment the receptors arepreferably those for growth factors, whereby preferably the growthfactors are selected from the group comprising PDGF, EGF and M-CSF, andalso comprise the nucleic acids coding therefor. In an embodiment thetranscription factors are selected from the group comprising YB-1, andalso comprise the nucleic acids coding therefor. In an embodiment themetalloproteinases are in particular matrix metalloproteinases. In apreferred embodiment the matrix metalloproteinases are selected from thegroup comprising MMP-1 and MMP-2, and also comprise the nucleic acidscoding therefor. In an embodiment the plasminogen activators of theurokinase type are selected from the group comprising uPa-R, and alsocomprise the nucleic acids coding therefor.

In a still further embodiment the medicament further comprises at leastone pharmaceutically active compound.

In a preferred embodiment the pharmaceutically active compound isselected from the group comprising cytokines, metalloproteinaseinhibitors, angiogenesis inhibitors, cytostatics such as Irinotecan andCPT-11 against colorectal carcinoma and Daunorubicin against leukemia,cell cycle inhibitors such as CYC202 which inhibits CDK2/CyclinE kinaseactivity and can be used against colorectal tumors (McClue S J, Int. J.Cancer 2002, 102, 463-468) and BAY 43-9006 which inhibits Raf-1 and is,for example, effective against mamma carcinoma (Wilhelm S M et al.,Cancer Res. 2004, 64, 7099-7109), proteosome inhibitors such as PS-341which inhibits the 26S proteasome activity and is used against braintumors, recombinant antibodies such as against the EGF receptor(Herceptin for breast carcinoma and prostate tumor; H. G. van der Poel,European Urology 2004, 1-17; Erbitux against head and neck tumors;Bauman M et al., Radiother. Oncol., 2004, 72, 257-266), and inhibitorsof the signal transduction cascade such as STI 571 which represses,among others, c-kit and can be used against gastrointestinal tumors (H.G. van der Poel, European Urology 2004, 45, 1-17), ABT-627, anendothelin inhibitor, which may be used, among others, against prostatetumors (H. G. van der Poel, European Urology 2004, 45, 1-17), SU5416which inhibits phosphorylation of the VEGF tyrosine kinase receptor andwhich may be used, among others, against neck and head tumors (Yin D. etal., Oncogene 2004), ZD1839 which inhibits EGFR tyrosine activity andmay be used, among others, against prostate tumors (H. G. van der Poel,European Urology 2004, 45, 1-17); rapamycin derivatives such as CCI-779and RAD001 which inhibit mTOR and can be used against prostate tumors.It is within the present invention that the various adenovirusesdescribed herein and the adenoviruses to be used in accordance with thepresent invention, respectively, can, in principle, be used with eachand any of the aforementioned compounds for each and any of theindication described herein in connection therewith. In a particularlypreferred embodiment the indication is the one which is described forany of the previously mentioned pharmaceutically active compounds.

In an embodiment the medicament comprises a combination of at least twoagents, whereby any agent is individually and independently selectedfrom the group comprising cytostatics.

In a preferred embodiment at least two of the agent act upon differenttarget molecules.

In an alternative embodiment at least two of the agents are activethrough a different mode of action.

In an embodiment at least one agents increases the capacity of a cell tobe infected in which the virus replicates.

In an embodiment at least one agent has an impact on the availability ofa component within the cell, which preferably increases the availabilityof the component, whereby the component mediates the uptake of thevirus.

In an embodiment at least one agent mediates the transport inof YB-1 tothe nucleus, preferably increases said transport.

In an embodiment at least one agent is a histone deacylase inhibitor.

In a preferred embodiment the histone deacylase inhibitor is selectedfrom the group comprising trichostatine A, FR 901228, MS-27-275,NVP-LAQ824 and PXD101.

In an embodiment at least one agent is selected from the groupcomprising trichostatin A, FR 901228 (against pancreas tumors, Sato N etal., Int. J. Oncol. 2004, 24, 679-685; MS-27-275 (against prostatetumors; Camphausen K et al., Clinical Canver Research 2004, 10,6066-6071), NVP-LAQ824 (against leukemiae; Nimmanapalli R et al., CancerRes. 2003, 63, 5126-5135; PXD101 (against ovary tumors, Plumb J A et al,Mol. Cancer Ther. 2003, 2, 721-728), Scriptaid (against breastcarcinoma, Keen J C et al., Breast Cancer Res. Treat. 2003, 81,177-186), apicidin (against melanoma, Kim S H et al., Biochem. Biophys.Res. Commun. 2004, 315, 964-970) and CI-994 (against various tumors,Nemunaitis J J et al., Cancer J. 2003, 9, 58-66). The mode of action ofhistone deacetylase inhibitors is, among others, described in LindemannR K et al., Cell Cycle 2004, 3, 77-86. It is within the presentinvention that the various adenoviruses described herein and theadenoviruses to be used in accordance with the present invention,respectively, may be used with the aforementioned compounds, inprinciple, for each and any of the indications described herein inconnection therewith. In a particularly preferred embodiment theindication is one as has been described for each and any of theaforementioned pharmaceutically active compounds.

In an embodiment at least one agent is a topoisomerase inhibitor.

In a preferred embodiment the topoisomerase inhibitor is selected fromthe group comprising camptothecin, irinotecan, topotecan, DX-895If,SN-38, 9-aminocamptothecin, 9-nitrocamptothecin, etoposid anddaunorubicin. These may be used against various tumors, for example,colorectal tumors, pancreas tumors, ovary carcinomas and prostatecarcinomas. The fields of use are, among others, described by Recchia Fet al., British J. Cancer 2004, 91, 1442-1446; Cantore M et al.,Oncology 2004, 67, 93-97; Maurel J. et al., Gynecol. Oncol 2004, 95,114-119; Amin A. et al., Urol. Oncol. 2004, 22, 398-403; Kindler H L etal., Invest. New Drugs 2004, 22, 323-327, Ahmad T. et al., Expert Opin.Pharmacother. 2004, 5, 2333-2340; Azzariti A. et al., Biochem Pharmacol.2004, 68, 135-144; Le Q T et al., Clinical Cancer Res. 2004, 10,5418-5424. It is within the present invention that the variousadenoviruses described herein and the adenoviruses to be used inaccordance with the present invention, respectively, may in principle beused with the aforementioned compounds for each and any of theindications described herein in connection therewith. In a particularlypreferred embodiment the indication is one as described for each of theaforementioned pharmaceutically active compounds.

In a preferred embodiment the agent comprises trichostatine A andirinotecan.

In an embodiment the virus, in particular the virus in accordance withone of the aspects of the present invention, is separated from the atleast two agents.

In a preferred embodiment at least one unit dosis of the virus isseparated from at least one unit dosis of one or the at least twoagents.

In a thirty-fourth aspect the invention is related to a kit comprising avirus, in particular a virus according to any aspect of the presentinvention, and at least two agents, whereby any agent is individuallyand independently selected from the group comprising cytostatics.

The above disclosed adenovirus according to the present invention,particularly those as described in connection with aspects one to eightof the present invention, are also referred to herein as group Iadenoviruses, and the adenoviruses having a transactivating oncogeneprotein such as, for example, E1A, and/or those referred to herein andparticularly above, as to be used in accordance with the presentinvention, are also referred to herein as group II adenoviruses. Group Iadenoviruses and group II adenoviruses are also collectively referred toherein as adenoviruses or adenoviruses according to the invention orviruses according to the invention.

The present invention is based on the surprising finding that thereversal of the expression sequence of adenoviral genes results in anefficient replication and optionally in the lysis of the cell infectedby the adenovirus. With regard to the chronologically changed expressionof the adenoviral genes particular emphasis is to be put on an E1Bprotein and an E4 protein which are also referred to herein,individually or collectively, as the first protein, which are expressedprior to a second protein. The second protein is selected from the groupcomprising E1A proteins. This expression sequence which is reversedcompared to wildtype adenoviruses where first an E1A protein and onlysubsequently the E1B protein and an E4 protein are expressed, ensuresthat transcription factors are activated, for example transported, intothe nucleus of the infected cell and influence the further replicationactivity or control the same there. The kinetics of the adenoviraltranscripts in wildtype adenoviruses are, for example, described inGlenn G. M. and Ricciardi R. P. Virus Research 1988, 9, 73-91, whoreport that in the wildtype the E1A transcripts, i.e. the E1A12Stranscript and the E1A13S transcript, are usually detectable prior tothe transcripts and translation products, respectively, E4orf6 andE1B55k. In the present case the E1B protein is, and also herein ingeneral if not indicated to the contrary, preferably the E1B-55 kDprotein. In the present case, the E4 protein is, and also herein ingeneral if not indicated to the contrary, preferably the E4orf6 protein.In the present case, the E1A protein is, and also herein in general ifnot indicated to the contrary, preferably an E1A12S protein or such anE1A protein as described herein in connection with the E1A-modifiedadenoviruses.

It is within the present invention that the E1A protein, in particularalso the E1A12S protein may be substituted in principle. Suchsubstituted E1A proteins and E1A12S proteins, respectively, are alsoreferred to herein as E1A protein and E1A12S protein, respectively, orshall be deemed to be comprised by this term, if not indicated to thecontrary. Instead of the E1A12S protein also an E1A protein may be usedwhich has a tumor suppressor function, such as, for example, describedby Dickopp A, Esche H, Swart G, Seeber S, Kirch H C, Opalka B. CancerGene Ther. 2000, July;7(7):1043-50. Further derivatives of E1A proteins,in particular of the E1A12S protein, as used and/or as referred to assuch herein, are generally also such proteins which are capable ofreleasing the factor E2F from the Rb/E2F complex. These are, amongothers, Simian virus 40 tumor antigen (SV40 large T antigen),papillomavirus E7 protein (HPV E7) as described by Chellappan S. et al.,Proc. Natl. Acad. Sci. USA 1992, 89, 4549-4533.

It is also within the present invention that derivatives of E4orf6 andE1B55k may be used, whereby the term E4orf6 and E1B55k, as used herein,comprises such derivatives. The derivatives are, for example, describedin Shen Y et al., J. of Virology 2001, 75, 4297-4307; Querido E. et al.,J. of Virology 2001, 75, 699-709.

It is within the present invention that an E1B protein is expressedprior to the E1A protein, or that an E4 protein is expressed prior to anE1A protein, or that both an E1B protein and an E4 protein are expressedprior to the E1A protein, each as described above.

An adenovirus designed in such a way is capable of replicating at aparticularly high level upon infection of a cell which expresses YB-1 inthe nucleus, preferably expresses YB-1 in the nucleus independent fromthe cell cycle, or which comprises deregulated YB-1, preferably in thecytoplasm. Without wishing to be bound thereto in the following thepresent inventor assumes that a complex consisting of E1B protein and/orE4 protein and individual ones of these two proteins, respectively,is/are capable of transporting deregulated YB-1 into the cellularnucleus or is/are capable of initiating adenoviral replication thereunder the influence of the E1B protein and/or E4 protein being expressedprior to the E1A protein. Once in the cellular nucleus or being presentthere in activated form, YB-1 may, as described herein, in particularusing the E2-late promoter, efficiently replicate. The chronologicallyearly expression of an E1B protein and/or an E4 protein thus avoids thecascade as observed in wildtype going along with initial expression ofE1A protein. In a preferred embodiment the E1A protein is an E1A proteinwhich is in particular no longer transactivating or transactivating onlyto a very limited extent the E1B protein and/or the E4 protein.Preferably, this transactivation is neither sufficient to ensure anefficient replication, nor sufficient to ensure replication in cellswhich do not have YB-1 in the nucleus. It is preferred that thetransactivation does not occur in cells which do not have YB-1 in thenucleus independent from the cell cycle or which do not have deregulatedYB-1.

Furthermore, the present invention is based on the surprising findingthat an adenovirus is capable of replicating in a particularly efficientmanner if it comprises at least a nucleic acid which codes for aprotein, whereby the protein is selected from the group comprising E1Bproteins, E4 proteins and E1A proteins and that at least one proteinthereof is under the control of a promoter which is different from thepromoter which controls the expression of the respective protein in awildtype adenovirus. Such replication is particularly efficient andusually results in tumor lysis in case the cells have YB-1 in thenucleus, in particular have YB-1 in the nucleus independent of the cellcycle, or in case the cells comprise deregulated YB-1, in particularcomprise deregulated YB-1 in the cytoplasm. What has been said aboveabout the E1B proteins, E4 proteins and E1A proteins applies also here.In wildtype adenoviruses the E1B protein is controlled by the E1Bpromoter, the E4 protein is controlled by the E4 promoter and the E1Aprotein is controlled by the E1A promoter. By selecting promoters whichare different from those which control the expression of theaforementioned proteins in wildtype adenoviruses, the expression of thepreviously mentioned proteins and thus the regulatory interplay of theindividual adenoviral nucleic acids and proteins is changed. Byselecting the promoters a chronologically different expression patterncan be created which, without wishing to be bound thereto in thefollowing, results in the observed replication in cells, whereby themechanism may be the one as already previously described with regard tothe chronologically different expression of the adenoviral proteins E1B,E4 and E1A. An example of a specific design for the control of saidproteins through promoters different from those controlling theexpression of the respective proteins in wildtype adenovirus, may betaken from the sub-claims and from the example part, whereby inparticular the viruses referred to therein as XVirPSJL1 and XVirPSJL2are representative thereof. Preferably, the E1B protein is the E1B55 kDprotein, the E4 protein is the E4orf6 protein and the E1A protein is theE1A12S protein.

The promoters which preferably control the E1B protein as well as the E4protein, are selected from the group comprising tumor-specificpromoters, organ-specific promoters, tissue-specific promoters,heterologous promoters and adenoviral promoters under the proviso thatwhen adenoviral promoters are used, they are different from the E1Bpromoter in case of the expression control of the E1B protein, and aredifferent from the E4 promoter in case of expression control of the E4protein. The use of the E1A promoter for the expression control of theE1B protein and/or the E4 protein is particularly preferred. The E1Apromoter is, for example, described by Boulanger P. A. and Blair, G. E.Biochem. J. 1991, 275, 281-299. Additionally, also the use of each andany other heterologous promoter is possible, i.e. a promoter which isdifferent from the one which controls the expression of the respectiveprotein in a wildtype adenovirus. A representative example is the CMVpromoter, whereby other promoters will be obvious for the ones skilledin the art.

The promoter which is used for the control of the E1A protein, may alsobe selected from the group comprising tumor-specific promoters,organ-specific promoters, tissue-specific promoters, heterologouspromoters and adenoviral promoters under the proviso that the adenoviralpromoter is different from the E1A promoter. It is within the presentinvention that one or several of the aforementioned proteins, i.e. theE1B protein, the E4 protein or the E1A protein are under the control ofthe same promoter, whereby it is nevertheless preferred thatparticularly the E1B protein and the E4 protein are under the control ofthe same promoter. It is particularly preferred that the expression ofthe E1A protein is controlled by a YB-1-controlled promoter or apromoter which can be regulated by YB-1. Such promoters are disclosedherein in connection with other aspects of the present invention. Theuse of the adenoviral E2-late promoter is particularly preferred for thecontrol of the expression of the E1A promoter as it can, first, beregulated by YB-1 and, second, shows only little transcription in theabsence of YB-1 which can factually be neglected so that a very goodexpression control of the nucleic acid which is under the control of theE2-late promoter, is ensured. This considerably increases biologicalsafety, particularly when applied in the field of medicine.

Furthermore, the present inventor has found that adenoviruses willreplicate particularly well in cells which have YB-1 in the nucleus,particularly have YB-1 in the nucleus independent of the cell cycle,and/or which have deregulated YB-1, preferably have deregulated YB-1 inthe cytoplasm, if YB-1 is provided for replication either directly orindirectly in particular in the cellular nucleus or if the provision ofYB-1 is directly or indirectly mediated through an adenoviral protein,whereby such adenoviral protein is different from E1A. This aspect ofthe present invention is different from the aspect which is alsodisclosed herein, namely that the use of transactivating E1A-modifiedadenoviruses, preferably group II adenoviruses, allows for replicationof these viruses in YB-1 nucleus-positive tumor cells, particularly YB-1nucleus-positive cells which are YB-1 positive independent of the cellcycle, and those cells which have deregulated YB-1, particularlycomprise YB-1 in the cytoplasm, insofar that the transactivatingcharacteristics of the E1A protein, particularly the E1A13S protein arenot used here, i.e. in connection with the group I adenoviruses, butrather in a preferred embodiment the E1A13S protein is functionallyinactive and is thus no longer capable of transactivating also E4orf6and E1B55k, which are involved in the transport and provision of YB-1,respectively, in the nucleus, either directly or indirectly.Consequently, an effective replication of the adenovirus is not possiblein accordance with this aspect of the present invention. Insofar, theprovision of YB-1 in the nucleus and the provision of YB-1 foradenoviral replication, respectively, is now no longer under the controlof the direct or indirect involvement of the E1A protein but occursthrough the expression of the E1B protein, particularly E1B55 kDprotein, and/or the E4 protein, particularly the E4orf6 protein, whichis not controlled by E1A.

This embodiment of the adenovirus may also be provided by one of theabove-described measures, for example by bringing forward thechronological expression of the E1B protein and/or the E4 proteincompared to the expression of the E1A protein, or by putting one orseveral of the E1B proteins, E4 proteins and E1A proteins under thecontrol of a promoter which is different from the promoter whichcontrols the expression of the respective protein in wildtypeadenovirus.

Finally, the present inventor starts from the surprising finding that aneffective adenoviral replication may also occur, particularly in cellswhich have YB-1 in the nucleus, more particularly YB-1 in the nucleusindependent of the cell cycle, or in cells which have deregulated YB-1,preferably in the cytoplasm, in case at least one of the E1B proteins,E4 proteins and E1A proteins, particularly the preferred forms thereof,are expressed in an expression cassette under the control of a promoter.In one embodiment of the present invention basically three expressioncassettes each comprising a single one of said proteins are provided. Inan alternative embodiment an expression cassette may also comprise twoor more of the proteins E1B, E4 and E1A and their derivatives andpossible substituents, respectively, particularly in case of E1A12S.What has previously been said in relation to the aspect that theadenoviruses comprise nucleic acids related to proteins E1B, E4 and E1A,is also applicable to the design of the various proteins and therespectively used promoters. When using such expression cassettes it ispreferred that proteins and nucleic acids coding therefor in the genomeof the wildtype adenovirus which correspond to the respective proteinsof the expression cassettes, are either completely or partially deletedto ensure that the virus is stable and to avoid recombinations, at leastto a bigger extent.

In principle, the expression cassettes can be cloned into each regionand each site, respectively, of the adenovirus, whereby preferably oneor several of the cassettes are inserted either individually or incombination with each other into the E1 region, the E3 region and/or theE4 region of the virus. It is possible that the nucleic acids of the E1,E3 and E4 region are completely deleted, partially deleted or notdeleted at all, whereby it is preferred with regard to the adenovirusesaccording to the invention that the nucleic acid coding for the E1A13Sgene is inactivated or deleted so as not to provide any transactivatingE1A protein by the virus. The extent of such deletion in one or severalof the regions E1, E3 and E4 is determined by the expression cassetteused and, optionally, further introduced foreign genes or transgenes orthe further expression cassettes comprising them, i.e. genes which aredifferent from the adenoviral genes, at least different in the sensethat they are not provided in the regulatory context of the adenoviralnucleic acid as prevailing in wildtype adenovirus or are not provided inthe sequence of the adenoviral nucleic acids of wildtype adenoviruses atsuch site. It is within the present invention that the nucleic acidswhich are contained in one or several of the expression cassettes whichcode for an E1B protein, an E4 protein and/or an E1A protein, arepartially or completely deleted in the adenoviral genome. In anembodiment, such as in the adenovirus according to the present inventionXvirPSJL1 or 2, the adenoviral nucleic acid coding for E4orf6 ispartially deleted and/or compeltely deleted, however, the completenucleic acid coding therefor is contained in the expression cassette.Preferably, this will also be realised for the E1B55k (also referred toas E155 Kd) protein and/or the E1A12S protein. The extent of thedeletion is to be selected in preferred embodiments such that a maximumpackage size of about 103% of the maximum package size of the wildtypeadenovirus is reached, although this limit is only a preferred limit.The possible deletions to be made in the adenoviral genome are onlysubject to limitations in preferred embodiments such as to make surethat still infectious and packed particles can be manufactured. Theprecise extent of the deletions may be determined by the ones skilled inthe art on the basis of the disclosure provided herein together withstandard tests.

As a starting point for the construction of the adenoviruses describedherein, any wildtype adenovirus may be used, but also other adenovirusesmay be used provided that they are constructed in accordance with thetechnical teaching of the present invention. It is particularlypreferred to have recourse to adenoviruses of subgroup C and within thisgroup in turn to adenovirus 2 and adenovirus 5.

The terms E1B protein and E1B proteins, E4 protein and E4 proteins aswell as E1A protein and E1 proteins are used herein in a synonymousmanner, if not indicated to the contrary.

As used herein, the term “deregulated” YB-1 refers to a YB-1 molecule orYB-1 protein as described herein which is present in a form which isquantitatively and/or qualitatively different from YB-1 as normallypresent in cells, preferably in non-tumor cells. A deregulated YB-1 canbe characterised and identified as such by particular viruses being ableto replicate in the presence of deregulated YB-1 in a cellularbackground comprising such deregulated YB-1. The particular viruses inconnection therewith are those the E1A protein of which is mutated andexhibits a transactivating function. Examples for these particularviruses are AD delta 24, dl 922-947, E1 Ad/01/07 and CB 016 and/or thosedescribed by Howe, J. A et al., Molecular Therapy 2, 485-495, 2000;Fueyo J. et al., Oncogene 19, 2-12, 2000; Heise C. et al., NatureMedicine 6, 1134-1139, 2001; Balague, C et al., J. Virol. 75, 7602-7611,2001; Bautista, D. S. et al., Virology 1991, 182, 578-596; Jelsma T. N.et al., Virology 1988, 163, 494-502; Wong, H. K. and Ziff E. B., J. ofVirology 1994, 68, 4910-4920]. Such a cell and a cell, respectively,having such a background can be used for the replication of group Iadenoviruses and/or group II adenoviruses. Additionally, tumorscomprising such cells may be lysed by the adenoviruses according to theinvention.

Furthermore, the present invention is based on the surprising findingthat the DNA replication of E1A-modified adenoviruses in YB-1nucleus-positive tumor cells is based on the activation of the E2-latepromoter. E1A-modified adenoviruses are to be understood as those which(a) have, in YB-1 nucleus-negative cells, a reduced or no replication atall compared to wildtype, (b) have a transactivation activity on atleast one viral gene, whereby the gene is particularly selected from thegroup comprising E1B-55 kDa, E4orf6, E4orf3 and E3ADP, and/or (c) do nottranslocate cellular YB-1 into the nucleus by the adenovirus.Optionally, the adenoviruses used in accordance with the presentinvention have the further characteristic that the binding of the E1Aprotein encoded by the adenovirus is interfering with the binding of E2Fto RB and is capable of dissolving the respective complex consisting ofE2F and Rb. Adenoviruses which have one or several of the aforementionedfeatures a) to c), preferably all of the features a) to c), arereplication deficient in cells which do not have YB-1 in the nucleus.

In an embodiment a strongly reduced replication herein in particularmeans a replication which is decreased compared to the wildtype by afactor of 2, preferably a factor of 5, more preferably a factor of 10and most preferably a factor of 100. In a preferred embodiment thecomparison of the replication is made using identical or similar celllines, identical or similar virus titres for the infection (multiplicityof infection, MOI or plaque forming unit, pfu) and/or identical orsimilar general experimental conditions. Replication particularly meansthe formation of particles. In further embodiments the measure forreplication may be the extent of viral nucleic acid synthesis. Methodsfor determining the extent of viral nucleic acid synthesis and methodsfor the determining particle formation are both known to the onesskilled in the art.

The findings, methods, uses or nucleic acids, proteins, replicationsystems and the like, are not necessarily limited to adenoviruses.Basically, such systems also exist in other viruses which are alsoencompassed herewith.

Using the viruses according to the present invention or the use of theviruses described herein in accordance with the present invention, mayresult in a replication comparable to wildtype when using an infectionrate of 1 to 10 pfu/cell compared to 10 to 100 pfu/cell in accordancewith the prior art.

Cellular YB-1 shall be any YB-1 which is encoded and is, preferably,also expressed by the cell, whereby this YB-1 is present in the cellparticularly prior to the infection of the respective cell by anadenovirus, preferably an adenovirus and/or a helper virus as describedherein. However, it is also within the present invention that cellularYB-1 is also a YB-1 which is introduced into the cell or produced by thecell only when exogenous measures such as infection with a virus,preferably an adenovirus, are applied.

Without wishing to be bound thereto, the present inventor assumes thatthe E2-early promoter, i.e. the early E2 promoter, is not switched on bymeans of the human cellular E2F transcription factor in connection withthe replication of the viruses used in accordance with the presentinvention and in connection with the use in accordance with the presentinvention of the adenoviruses of the present invention. Under suchcircumstances the start of the replication is independent of the Rbstatus of the cells, i.e. the tumor cells which are infected by usingthe viruses disclosed herein and which are preferably lysedsubsequently, may contain either functional as well as inactive Rbproteins. In addition, adenoviral replication using the adenovirusesdisclosed herein or using the conditions disclosed herein, does notrequire any functional p53 protein, however is neither negativelyaffected by its presence. Insofar the technical teaching turns away fromthe principle underlying the use of oncolytic or tumorlytic adenovirusesof the type of AdΔ24, dl922-947, E1Ad/01/07, CB016 or those adenovirusesdescribed, for example, in European patent EP 0 931 830, which had beenmade subject to one and/or several deletion(s) in the E1A protein underthe assumption that intact functional Rb proteins would hinder anefficient in vivo replication and thus provide for adenoviralreplication in vivo only in Rb-negative and Rb-mutated cells. Theseadenoviral systems of the prior art are based on E1A in order to controlin vivo replication of adenoviruses by means of the early E2 promoter(E2-early promoter) and “free E2F”. Nevertheless, these known viruses ofthe prior art may be used in accordance with the present invention forthe replication in cells which contain YB-1 in the nucleus independentof the cell cycle, or in cells which comprise deregulated YB-1.

The viruses in particular adenoviruses described in said European patentEP 0 931 830 may be used in accordance with the present invention. Morespecifically, the viruses described in said patent are viruses which arereplication deficient and which lack an expressed viral oncoproteinwhich is capable of binding a functional Rb tumor suppressor geneproduct. The adenovirus can particularly be any adenovirus which lacksexpressed viral E1A oncoprotein which is capable of binding a functionaltumor suppressor gene product, more particularly Rb. The viral E1Aoncoprotein can exhibit an inactivating mutation, for example in the CR1domain at the amino acid positions 30 to 85 in adenovirus Ad5, which isalso referred to herein as Ad5, Ad 5, the nucleotide positions 697-790and/or the CR2 domain at amino acid positions 120 to 130 in Ad 5, thenucleotide position 920 to 967 which are involved in the binding of p105Rb protein, p130 and p107 protein. However, it is within the presentinvention that the adenovirus is of type 2 dl 312 or type 5 NT dl 1010.

In connection with the use of adenoviruses in accordance with thepresent invention for the manufacture of a medicament, in particular forthe manufacture of a medicament for the treatment of tumor diseases andof the other diseases disclosed herein, and in connection with the useof adenoviruses in accordance with the present invention as well as theuse of the adenoviruses according to the present invention forreplication in cells which have YB-1 in the nucleus, preferably haveYB-1 in the nucleus independent of the cell cycle or which comprisederegulated YB-1, preferably in the cytoplasm, replication finallyoccurs in those cells which have YB-1 in the nucleus, preferablyindependent of the cell cycle, which are, in other words, YB-1nucleus-positive, or in cells which comprise deregulated YB-1. It isparticularly to be acknowledged that the adenoviruses as such do notreplicate or only replicate at a significantly reduced level in cellswhich do not have YB-1 in the nucleus but only contain YB-1 in thecytoplasm, or in cells which do not contain any deregulated YB-1.Insofar it is necessary for a successful replication of these virusesthat YB-1 is present in the nucleus, preferably independent of the cellcycle, or that deregulated YB-1 is present. As will also be explained inthe following, this can be achieved, for example, by applying to thecells conditions which result in the expression or presence of YB-1,preferably independent of the cell cycle, or deregulated YB-1 in thenucleus or in the expression of deregulated YB-1. A respective measurecan, for example, be the coding and expression, respectively, of YB-1 bythe adenoviruses which are either used in accordance with the presentinvention or which are subject to the present invention, which inaddition to the adenoviral genes also carry genetic information whichcodes for YB-1 and which particularly codes for its expression. Othermeasures which result in the transport, induction or expression of YB-1in the nucleus of the cell, are application of stress such as theadministration to the cell and to an organism containing such a cell ofcytostatics, irradiation, hyperthermia and the like. In a preferredembodiment irradiation is any radiation which is, for example, used inthe treatment of tumor diseases.

The adenoviruses used in accordance with the present invention,particularly for tumor lysis, as well as the adenoviruses according tothe invention are characterised in preferred embodiments by the factthat they do not replicate in cells which do not have YB-1 in thenucleus independent of the cell cycle and which are thus YB-1nucleus-negative, or which do not comprise any deregulated YB-1.

A further feature of a part of the adenoviruses to be used in accordancewith the present invention which are different from the adenoviruses ofthe present invention, is that they code for a viral oncogene which isalso referred to herein as oncogene protein, whereby the oncogeneprotein is preferably E1A and whereby the oncogene protein is capable ofactivating at least one viral gene which has an impact on thereplication of the virus and/or cell lysis of the cell infected by saidvirus. Preferably, the impact on the replication is such that the virusreplicates better in the presence of the oncogene protein compared tothe scenario where the oncogene protein of the respective virus isabsent. This process is also referred to herein as transactivating andparticularly as E1A transactivating in case the transactivation ismediated by E1A. The term “transactivate” or “transactivation”preferably describes the process that the respective viral oncoproteinhas an impact on the expression and/or on the transcription of one orseveral other genes which are different from the gene coding for theviral oncogene protein itself, i.e. controls its/their expression and/ortranslation and particularly activates it/them. Such viral genes arepreferably E1B55 kDa, E4orf6, E4orf3 and E3ADP as well as anycombination of the aforementioned genes and gene products, respectively.

A further, although only optional feature of the adenoviruses to be usedin accordance with the present invention as well as of the adenovirusesof the present invention is their binding characteristics and thebinding characteristics of particular ones of the proteins coded bythem, respectively, to tumor suppressor Rb. Basically, it is within thepresent invention that the adenoviruses used in accordance with thepresent invention may or may not bind to Rb. The use of any of the twoalternative embodiments of the adenoviruses is independent of the Rbstatus of the cells treated or the cells to be treated.

In order to confer to E1A the ability not to bind to Rb, the followingdeletions can be made to the E1A oncoprotein: deletion in the CR1 region(amino acid positions 30-85 in Ad5) and deletion of the CR2 region(amino acid positions 120-139 in Ad5). In doing so, the CR3 region ispreserved and can exercise its transactivating function on the otherearly viral genes.

In order to confer to E1A the ability to bind to Rb, the followingdeletions to E1A oncoprotein, however, are basically possible: deletionof the CR3 region (amino acid positions 140-185); deletion of theN-terminus (amino acid positions 1-29); deletion of the amino acidpositions 85-119; and deletion of the C-terminus (amino acid positions186-289). The regions listed above do not interfere with the binding ofE2F to Rb. The transactivating function remains intact, however, isreduced compared to wildtype Ad5.

It is also within the present invention, particularly with regard to theadenoviruses of the present invention, that the E1A protein,particularly the E1A12S protein is designed such that, in an embodiment,it is capable of binding to Rb and, in a different embodiment, is notcapable of binding to Rb, whereby such E1A12S protein is an E1A proteinand particularly an E1A12S protein in the meaning of the presentinvention which is nevertheless referred to in the prior art sometimesas modified E1A12S. The respective design of the E1A12S protein iswithin the skills of those of the art, particularly with regard to theaforementioned deletions of the E1A protein which is also referred toherein simply as E1A.

Such adenoviruses which are basically already known in the prior art andwhich do not show any transactivation, are generally regarded asreplication deficient. However, it is the merit of the present inventorthat he has recognised that they are nevertheless capable of replicatingin a suitable background, in particular a cellular background. Suchsuitable cellular background is caused or provided by the presence ofYB-1 in the nucleus, preferably a cell cycle independent presence ofYB-1 in the nucleus, or by deregulated YB-1. The term cells or cellularsystems as used herein in connection with each and any other aspect ofthe present invention, comprises fragments or fractions of cell extractsas well as cells which are present in vitro, in vivo or in situ.Insofar, the term cellular systems or cells also comprises cells whichare present in cell culture, tissue culture, organ culture or in anytissue or organ in vivo and in situ, respectively, isolated, in groupsor as part of tissues, organs or organisms, but which may also bepresent as such in a preferably living organism. The organism ispreferably any vertebrate organism and more preferably a mammal. Morepreferably the organism is a human organism. Other preferred organismsare those disclosed in connection with the various aspects of thepresent invention.

Additionally, it is within the present invention that based on thetechnical teaching provided herein, new viruses are generated which showthe replication behaviour of the adenoviruses described herein and ofthose of the prior art in such cells which are YB-1 nucleus-positive,preferably YB-1 nucleus-positive independent of the cell cycle, or whichcomprise deregulated YB-1. In other words, particularly startingpreferably from the adenoviruses already known, further viruses can beconstructed which exhibit the features defined herein which are relevantfor the use in accordance with the invention.

In connection with the present invention the modified E1A oncoprotein ofthe various adenoviruses to be used in accordance with the presentinvention is, in contrast to the viruses of the present invention,capable of transactivating the early viral genes such as E1B55K, E4orf3, E4orf6, E3ADP in YB-1 nucleus-positive cells or cells whichcomprise deregulated YB-1. There are preferably no other changes made tothe viral genome and the respective adenovirus may insofar correspondotherwise to a wildtype adenovirus or a derivative thereof.

The viruses disclosed herein which code or comprise a transactivatingoncogene protein in the meaning of the present invention, comprise, forexample, the adenoviruses AdΔ24, dl922-947, E1Ad/01/07, CB106 and/or theadenoviruses described in European patent EP 0 931 830 which are eachcapable of transactivating the early genes such as E1B, E2, E3 and/or E4and which are comparable to the adenoviruses of wildtype, particularlywildtype Ad5. In these cases, a distinct region of the E1A protein isresponsible for the transactivation. Within the various adenoviralserotypes there are three highly conserved regions within the E1Aprotein. The region CR1 from amino acid positions 41-80, CR2 from aminoacid positions 120-139 and CR3 from amino acid positions 140-188. Thetransactivating function is mainly based on the presence of the CR3region within the E1A protein. The amino acid sequence of CR3 is presentin an unchanged manner in the above mentioned adenoviruses. This resultsin a transactivation of the early genes E1B, E2, E3 and E4 independentof whether YB-1 is present in the nucleus or in the cytoplasm.

In contrast thereto, the CR3 region has been deleted in the recombinantadenovirus dl520. Thus, dl520 expresses a so-called E1A12S protein whichdoes not comprise the amino acid sequence of the CR3 region.Consequently, dl520 may exercise only a very weak transactivatingfunction, particularly on the E2 region, and thus does not replicate inYB-1 nucleus-negative cells. In YB-1 nucleus-positive cells YB-1 isresponsible for the transactivation of the E2 region and thus allows foran efficient replication of dl520. The use of systems like dl520 orsystems originating therefrom for the purposes disclosed herein, isbased thereon. A further important difference between the two previouslydescribed groups of adenoviruses such as, for example, delta 24 (alsoreferred to herein as AdΔ24) and, for example, dl520, resides in thefact that the early genes E1B, E3 and E4 are more comprehensivelytransactivated in cells being YB-1 nucleus-positive cells independent ofthe cell cycle or in cells containing deregulated YB-1, compared to YB-1nucleus-negative cells or cells which do not comprise deregulated YB-1.In contrast thereto, there are no or only minor differences in delta 24.The transactivation of dl520, more specifically of the E1A12S proteinis, however, significantly reduced compared to wildype adenovirus. Thistransactivation, however, is sufficient so as to provide for anefficient replication in YB-1 nucleus-positive cells as also shown inexample 10. The design of the E1A protein as described herein and inparticular as described in this connection, and of the nucleic acidcoding therefor, such that the E1A protein has, compared to the wildtypeoncogene protein E1A, one or several deletions and/or mutations,including and particularly preferably those designs of the E1A proteinas described in connection with dl520 or AdΔ24, dl922 to 947,E1Ad/01/07, CB106 and/or the adenoviruses described in European patentEP 0 931 830, are embodiments of viruses, in particular of adenoviruses,the replication of which is controlled, preferably predominantlycontrolled by the activation of the E2-late promoter. Preferably, thedeletion is such that it is selected from the group comprising deletionsof the CR3 region and deletions of the N-terminus and deletions of theC-terminus. Further embodiments of the E1A protein which allow this kindof replication of adenoviruses, can be generated by the ones skilled inthe art based on the disclosure provided herein. The embodiment of theE1A protein as described previously is an embodiment which may also beused in connection with the adenoviruses of the present invention whichare also referred to herein as adenoviruses of the present invention orgroup I adenoviruses.

The adenoviruses of the present invention, particularly the group Iadenoviruses, which are also referred to herein as derivatives and whichmay be used in accordance with the present invention, typically comprisean E1 deletion, an E1/E3 deletion and/or an E4 deletion, i.e. thecorresponding adenoviruses are not capable of generating functionallyactive E1 and/or E3 and/or E4 expression products and correspondingproducts, respectively. Or in other words these adenoviruses are onlycapable of generating functionally inactive E1, E3 and/or E4 expressionproducts, whereby a functionally inactive E1, E3 and/or E4 expressionproduct is an expression product which is either not present as anexpression product at all, either at the transcription level and/or atthe translation level, or is present in a form which at least does nothave one of the functions attributed to it in a wildtype adenovirus.This/these function(s) inherent to the expression product in wildtypeadenovirus is/are known to the ones skilled in the art and, for example,described in Russell, W. C., Journal of Virology, 81, 2573-2604, 2000.Russell (supra) also describes design principles of adenoviruses andadenoviral vectors which are incorporated herein by reference. It isalso within the present invention that the modified E1A oncoprotein,i.e. the no longer transactivating E1A protein and other proteins suchas E1A12S, E1B-55K, E4orf6 and/or E3ADP (adenoviral death protein (ADP))(Tollefson, A. et al., J. Virology, 70, 2296-2306, 1996) are expressedin such vector either alone or in any combination. The individualmentioned genes as well as the transgenes disclosed herein, may be,independently from each other, cloned into the E1 and/or E3 and/or E4region and expressed using a suitable promoter or under the control of asuitable promoter. Basically, each of the E1, E3 and E4 region issuitable as cloning site within the adenoviral nucleic acid, whereby theregions not used for the cloning can, either individually or as a whole,be present, partially and/or completely deleted. In case that theseregions are present, in particular are present in their entirety, it iswithin the present invention that they are either intact and preferablyprovide for a translation product and/or a transcription product, and/orare not intact and preferably do not provide for a translation productand/transcription product. In some embodiments suitable promoters arethose as disclosed herein in connection with the control and expression,respectively, of E1A, preferably of the modified E1A.

Finally, in an embodiment, the group II adenoviruses used in accordancewith the present invention are E1B deficient, particularly E1B 19 kDadeficient. The term deficient as generally used herein refers to acondition, wherein the E1B does not exhibit all of the characteristicsof the wildtype E1B and lacks at least one of these characteristics.

The adenoviral BCL2 homolog E1B 19k avoids the E1A induced apoptosis byinteraction with the pro-apoptotic proteins Bak and Bax. Because of thisa maximum replication and/or particle formation is possible in infectedcells (Ramya Sundararajan and Eileen White, Journal of Virology 2001,75, 7506-7516). The lack of E1B19k results in a better release of theviruses as, if present, it will minimize the function of the adenoviraldeath protein. By such a deletion the virus induced cytopathic effect isincreased (Ta-Chiang Liu et al., Molecular Therapy, 2004) and thusresults in a more pronounced lysis of infected tumor cells.Additionally, the lack of E1B19k causes that TNF-alpha does not exert aninfluence on the replication of such recombinant adenoviruses in tumorcells, whereas in normal cells the treatment results in a reducedreplication and release of infectious viruses. Insofar, selectivity andspecificity are increased (Ta-Chiang Liu et al., Molecular Therapy 2004,9, 786-803).

At least some embodiments of the group II adenoviruses as used inaccordance with the invention disclosed herein, are as such known in theart. The adenoviruses used in accordance with the invention arepreferably recombinant adenoviruses, particularly also if, compared tothe wildtype, a change has been made in the sense of the technicalteaching provided herein. It is within the skills of those of the art todelete and mutate, respectively, the adenoviral nucleic acid sequenceswhich are irrelevant for the invention. Such deletions may be relatedto, e.g. a part of the E3 and E4 coding nucleic acids as also describedherein. A deletion of E4 is particularly preferred provided that suchdeletion does not extend to the protein E4orf6, in other words theadenovirus to be used in accordance with the invention codes for E4orf6.In preferred embodiments, these adenoviral nucleic acids may still bepacked into viral capsids and thus form infectious particles. This isalso true for the use of the nucleic acids in accordance with theinvention. Generally it is also to be acknowledged that the adenoviralsystems may be deficient with regard to single or several expressionproducts. In connection therewith it is to be taken into considerationthat this, in connection with both the group I adenoviruses and thegroup II adenoviruses, may be caused by the mutation or deletion of thenucleic acid coding the expression product, whereby such mutation anddeletion, respectively, is either a complete one or performed to theextent that no expression product is formed anymore or by the regulatoryelements and elements controlling the expression such as promoters andtranscription factors being missing or being active in a way differentfrom wildtype, either at the level of the nucleic acid (lack of apromoter; cis acting elements) or at the level of the translation andtranscription system (transacting elements), respectively. Particularlythe latter aspect may depend on the respective cellular background.

Apart from using adenoviruses which are as such already known, inaccordance with the present invention also novel adenoviruses such asgroup II adenoviruses may be used for the purposes already disclosed forthe other adenoviruses described herein. The new adenoviruses of theinvention result from the technical teaching provided herein.Particularly preferred representatives are, for example, the virusesXvir03 and Xvir03/01 which are depicted in FIGS. 16 and 17, the designprinciple of which is further illustrated in examples 11 and 12.

In case of vector Xvir03 a CMV promoter was cloned into the E1 regionwhich controls the nucleic acids for E1B 55k and E4orf6 which areseparated by an IRES sequence. In connection therewith the E3 and E4region may be deleted and/or may be present in an intact form Due to thecloning of these two genes into the virus and due to the gene productsgenerated therefrom, respectively, a high replication efficiencyresults, whereby the selective replication in cells, preferably tumorcells, is maintained insofar as a replication occurs particularly inYB-1 nucleus-positive cells and more particularly in those cells whichcomprise deregulated YB-1 in the sense of the present disclosure. Cellsin which deregulated YB-1 is present are, in an embodiment, cells whichshow an increased expression of YB-1, preferably compartment independentexpression of YB-1, compared to normal or non-tumor cells. E1B 55k andE4orf6 can also be cloned into the E4 region, whereby the E3 region canbe intact or/and partially or completely deleted.

A further development of virus Xvir03 is virus Xvir03/01 into which in apreferred embodiment therapeutic genes or transgenes have been clonedunder the control of a specific promoter, in particular a tumor-specificor tissue-specific promoter. In connecton therewith the E3 and the E4region can be deleted and/or be intact. Furthermore, in connection withsuch virus also the E4 region is functionally inactive, is preferablydeleted. The transgenes described herein may also be cloned into the E4region, whereby this can be done either alternatively or in addition tothe cloning of the transgenes into the E3 region.

The transgenes described herein and particularly described in thefollowing, may also be expressed in connection with or by theadenoviruses of the present invention, i.e. group I adenoviruses andtheir nucleic acids, respectively, or the replication systems of theinvention and are thus comprised in connection with an expressioncassette comprising a promoter and a nucleic acid sequence, whereby suchnucleic acid sequence codes for one or several of said transgenes. TheE1, E3 and/or E4 regions are particularly suitable cloning sites in theadenoviral genome, however, the cloning sites are not limited thereto.Transgenes as used herein, may be viral genes, preferably adenoviralgenes, which are preferably not present in the genome and, respectively,which are not present at the site of the genome of the wildtype wherethey are present in the particular virus now, or therapeutic genes.

Therapeutic genes may be prodrug genes, genes for cytokines, apoptosisinducing genes, tumor suppressor genes, genes for metalloproteinaseinhibitors and/or angiogenesis inhibitors, and tyrosine kinaseinhibitors. Additionally, siRNA, aptamers, antisense molecules andribozymes may be expressed which are preferably directed againstcancer-relevant target molecules. Preferably the individual or theseveral target molecules are selected from the group comprising theresistance-relevant factors, anti-apoptosis factors, oncogenes,angiogenesis factors, DNA synthesis enzymes, DNA repair enzymes, growthfactors and their receptors, transcription factors, metalloproteinases,particularly matrix metalloproteinases, and plasminogen activator of theurokinase type. Preferred embodiments thereof are those which have beendisclosed already herein in connection with other aspects of theinvention.

Possible prodrug genes as may be used in preferred embodiments, are, forexample, cytosine deaminase, thymidine kinase, carboxypeptidase, uracilphosphoribosyl transferase; or purine nucleoside phosphorylase (PNP);[Kim et al, Trends. in Molecular Medicine, volume 8, no. 4 (suppl),2002; Wybranietz W. A. et al., Gene Therapy, 8, 1654-1664, 2001;Niculescu-Duvaz et al., Curr. Opin. Mol. Therapy, 1, 480.486, 1999;Koyama et al., Cancer Gene Therapy, 7, 1015-1022, 2000; Rogers et al.,Human Gene Therapy, 7, 2235-2245, 1996; Lockett et al., Clinical CancerRes., 3, 2075-2080, 1997; Vijayakrishna et al., J. Pharmacol. And Exp.Therapeutics, 304, 1280-1284, 2003].

Possible cytokines as may be used in preferred embodiments, are, forexample, GM-CSF, TNF-alpha, Il-12, Il-2, Il-6, CSF or interferon-gamma;[Gene Therapy, Advances in Pharmacology, volume 40, editor: J. ThomasAugust, Academic Press; Zhang and Degroot, Endocrinology, 144,1393-1398, 2003; Descamps et al., J. Mol. Med., 74, 183-189, 1996;Majumdar et al., Cancer Gene Therapy, 7, 1086-1099, 2000].

Possible apoptosis-inducing genes as may be used in preferredembodiments, are, for example, Decorin [Tralhao et al., FASEB J, 17,464-466, 2003]; retinoblastoma 94 [Zhang et al., Cancer Res., 63,760-765, 2003]; Bax and Bad [Zhang et al., Hum. Gene Ther., 20,2051-2064, 2002]; apoptin [Noteborn and Pietersen, Adv. Exp. Med. Biol.,465, 153-161, 2000]; ADP [Toth et al., Cancer Gene Therapy, 10, 193-200,2003]; bcl-xs [Sumantran et al., Cancer Res, 55, 2507-2512, 1995];E4orf4 [Braithwaite and Russell, Apoptosis, 6, 359-370, 2001]; FasL,Apo-1 and Trail [Boehringer Manheim, Guide to Apoptotic Pathways, Araiet al., PNAC, 94, 13862-13867, 1997]; Bims [Yamaguchi et al., GeneTherapy, 10, 375-385, 2003; GNR163: Oncology News, 17 Jun., 2000].

Possible tumor suppressor genes as may be used in preferred embodiments,are, for example, E1A, p53, p16, p21, p27 or MDA-7 [Opalka et al., CellTissues Organs, 172, 126-132, 2002, Ji et al., Cancer Res., 59,3333-3339, 1999, Su et al., Oncogene, 22, 1164-1180, 2003].

Possible angiogenesis inhibitors as may be used in preferredembodiments, are, for example, endostatin or angiostatin [Hajitou etal., FASEB J., 16, 1802-1804, 2002], and antibodies against VEGF[Ferrara, N., Semin Oncol 2002 December; 29 (6 suppl 16): 10-4].

Possible metalloproteinase inhibitors as may be used in preferredembodiments, are, for example, Timp-3 [Ahonen et al., Mol Therapy, 5,705-715, 2002]; PAI-1 [Soff et al., J. Clin. Invest., 96, 2593-2600,1995]; Timp-1 [Brandt K. Curr. Gene Therapy, 2, 255-271, 2002].

Further transgenes in the sense of the present invention which may beexpressed by both group I adenoviruses and group II adenoviruses arealso tyrosine kinase inhibitors. Exemplary tyrosine kinases are EGFR(epidermal growth factor receptor) [Onkologie, Entstehung undProgression maligner Tumoren; author: Christoph Wagner, Georg ThiemeVerlag, Stuttgart, 1999]. A preferred tyrosine kinase inhibitor isherceptin [Zhang H et al., Cancer Biol Ther. 2003, July-August; 2 (4suppl 1): S122-6].

SiRNA (short interfering RNA) which may be used in connection with thepresent invention, consists of two, preferably separate RNA strandswhich hybridise to each other due to base complementarity which meansthat they are present essentially base paired and preferably have alength of up to 50 nucleotides, preferably between 18 and 30nucleotides, more preferably less than 25 nucleotides and mostpreferably 21, 22 or 23 nucleotides, whereby these figures refer to thesingle strand of the siRNA, particularly to the length of the stretch ofthe single strand which hybridises to or is base paired with a, moreprecisely the second single strand. siRNA specifically induces ormediates the degradation of mRNA. The specificity required theretoforeis mediated by the sequence of the siRNA and thus its binding site. Thetarget sequence to be degraded is essentially complementary to the firstor to the second of the siRNA forming strands. Although the precise modeof action is not yet clear, it is assumed that siRNA is a biologicalstrategy for cells in order to inhibit distinct alleles duringdevelopment and to protect themselves against viruses. siRNA mediatedRNA interference is used as a method for the specific suppression orcomplete elimination of the expression of a protein by introducing agene specific double-stranded RNA. For higher organisms a siRNAcomprising 19 to 23 nucleotides is insofar particularly suitable as itdoes not result in the activation of a non-specific defense reactionsuch as an interleukin response. The direct transfection ofdouble-stranded RNA of 21 nucleotides having symmetrical 2-nt 3′overhangs was suitable to mediate RNA interference in mammalian cellsand is highly efficient compared to other technologies such as ribozymesand antisense molecules (Elbashir, S. Harborth J. Lendeckel W. Yalvcin,A. Weber K, Tuschl T: Duplexes of 21-nucleotide RNAs mediate RNAinterference in cultured mammalian cells. Nature 2001, 411: 494-498). Aslittle as a few siRNA molecules are sufficient so as to suppressexpression of the target gene. In order to avoid the limitations ofexogenously added siRNA which particularly reside in the transientnature of the interference phenomenon and specific delivery (delivery)of the siRNA molecules, vectors are used in the prior art which allowfor an endogenous siRNA expression. For such purpose, for example,oligonucleotides having a length of 64 nucleotides are introduced intothe vector which comprise the 19 nucleotide long target sequence both inthe sense and in the antisense orientation, separated by, for example, a9 nucleotide spacer sequence. The resulting transcript folds into ahairpin structure with a stem structure (stem) of, for example, 19 basepairs. The loop is rapidly degraded in the cell so that a functionalsiRNA molecule is generated (Brummelkamp et al., Science, 296, 550-553,2002).

The nucleic acid coding for YB-1 which may be part of the adenovirusesin an embodiment of the adenoviruses to be used in accordance with theinvention, particularly group II adenoviruses, but also of theadenoviruses according to the invention, i.e. group I adenoviruses, maycomprise a nucleic acid sequence which mediates the transport of YB-1into the nucleus. The nucleic acids, adenoviruses and adenoviral systemsaccording to the invention as well as the adenoviruses known in theprior art such as, for example, Onyx-15, AdΔ24, dl922-947, E1Ad/01/07,CB016, dl 520 and the adenoviruses described in patent EP 0 931 830 maybe used, as adenoviruses and adenoviral systems, respectively, and thecorresponding nucleic acids, in combination with these nucleic acids inaccordance with the invention. Suitable nucleic acid sequences mediatingnuclear transport are known to the ones skilled in the art and, forexample, described in Whittaker, G. R. et al., Virology, 246, 1-23,1998; Friedberg, E. C., TIBS 17, 347, 1992; Jans, D. A. et al.,Bioassays 2000 June; 22(6): 532-44; Yoneda, Y., J. Biochem. (Tokyo) 1997May; 121(5): 811-7; Boulikas, T., Crit. Rev. Eukaryot. Gene Expr. 1993;3(3): 193-227; Lyons R H, Mol. Cell Biol., 7 2451-2456, 1987). Thenucleic acid sequences mediating nuclear transport may realise differentprinciples. One such principle is that YB-1 forms a fusion protein witha signal peptide or is provided with such signal peptide and istransferred into the cellular nucleus because of the signal peptide,whereupon the replication of the adenoviruses in accordance with theinvention occurs.

A further principle which may be used in the design of the adenovirusesto be used in accordance with the invention, particularly group IIadenoviruses, but also with the adenoviruses in accordance with thepresent invention, i.e. the group I adenoviruses, is providing YB-1 witha transport sequence which results in the transfer or translocation ofYB-1 into the cellular nucleus, preferably starting from a synthesis inthe cytoplasm, and prompts viral replication there. An example for aparticularly effective nucleic acid sequence mediating transport intothe nucleus, is the TAT sequence of HIV which is, for example, describedtogether with other suitable nucleic acid sequences of that kind inEfthymiadis, A., Briggs, L J, Jans, D A., JBC 273, 1623-1628, 1998. Itis within the present invention that the adenoviruses to be used inaccordance with the invention, particularly group II adenoviruses, butalso the adenoviruses according to the present invention, i.e. group Iadenoviruses, comprise the nucleic acid sequences which code for thepeptides which mediate nuclear transport.

It is within the present invention that YB-1 is present in its fulllength, particularly in a form which corresponds to wildtype YB-1.Furthermore, it is within the invention that YB-1 is used or present asa derivative, for example in a shortened or truncated form. A YB-1derivative as may be used or may be present in connection with thepresent invention, is a YB-1 which is preferably capable of binding tothe E2 late promoter and thus activates gene expression of theadenoviral E2 region. Such derivatives particularly comprise the YB-1derivatives disclosed herein. Further derivatives can be generated bydeletion of single or several amino acids at the N-terminus, theC-terminus or within the amino acid sequence. It is within the presentinvention that also YB-1 fragments are used as YB-1 proteins in thesense of the present invention. In the paper of Jürchott K et al. [JBC2003, 278, 27988-27996] various YB-1 fragments are disclosed which arecharacterised by deletions at the C- and the N-terminus. Thedistribution of the various YB-1 fragments has shown that both the coldshock domain (CSD) as well as the C-terminus is relevant for the cellcycle regulated transport of YB-1 into the cellular nucleus. It is thuswithin the present invention that a shortened YB-1 (herein also referredto as YB-1 protein) in connection with the inventive expression ofE1B55k and E4orf6 migrates better into the nucleus and thus induces astronger CPE without necessarily binding better to the E2-late promotercompared to native YB-1, whereby it cannot be excluded that also ashortened YB-1 migrates better into the nucleus and is causing botheffects, i.e. induces CPE and binds to the E2-late promoter. Finally,such shortened YB-1 fragments may also migrate better into the nucleusand bind more efficiently to the E2-late promoter without inducing abetter CPE. It is also within the present invention that shortened YB-1proteins and fragments, respectively, comprise further sequences asdisclosed herein in connection with the full length YB-1, in particularcell localisation signal sequences (NLS) and the like.

With regard to the aforementioned various further genes and geneproducts encoded and expressed, respectively, by the adenovirus, it isin principle possible that these are coded and expressed, respectively,in any combination.

It is within the present invention that the terms adenovirus andadenoviral systems are to be understood as having essentially the samemeaning. The term adenovirus shall particularly be understood such as tobe related to the complete virus particle comprising the capsid and thenucleic acid. The term adenoviral system particularly focuses on thefact that the nucleic acid is changed compared to the wildtype.Preferably such changes comprise changes in the set-up of the genome ofthe adenovirus as may result from deleting and/or adding and/or mutatingpromoters, regulatory sequences and/or coding sequences such as readingframes. The term adenoviral system is additionally more preferably usedsuch that it is a vector which may, for example, be used in genetherapy.

The above comments, including any use and any design of the adenovirusesand adenoviral systems, respectively, are also applicable to the nucleicacids coding therefor and vice versa.

In connection with the present invention it is possible that theadenoviruses used in accordance with the invention, particularly groupII adenoviruses, but also group I adenoviruses and the nucleic acidscoding therefor, is any respective adenoviral nucleic acid which as suchor in combination with further nucleic acid sequences results in areplication event. It is possible, as explained herein, that thesequences and/or gene products necessary for replication are provided byhelper viruses. To the extent it is referred to coding nucleic acidsequences and said nucleic sequences are nucleic sequences which areknown, it is within the present invention that not only the identicalsequence is used but also sequences derived therefrom. Herein, derivedsequences shall mean in particular any sequences which still result in agene product, either a nucleic acid or a polypeptide which has afunction which corresponds to a or the function of the non-derivedsequence. This can be tested by routine tests known to the one skilledin the art. An example for such derived nucleic acid sequences are thosenucleic acid sequences which code for the same gene product, inparticular for the same amino acid sequence, which, however, have adifferent base sequence due to the degeneracy of the genetic code.

With regard to the adenoviruses according to the invention of group IIand/or the corresponding adenoviral replication system according to theinvention and their use in accordance with the invention, respectively,in an embodiment the adenoviral nucleic acid is deficient for theexpression of the oncogene protein, in particular is E1A proteindeficient, i.e. does either not code for the 12S E1A protein (hereinalso referred to as E1A12S protein) or for the 13S E1A protein (hereinalso referred to as E1A13S protein) or does not code for both the 12SE1A protein and the 13S E1A protein, or is modified, as defined herein,if not indicated to the contrary, and that the adenoviral replicationsystem further comprises a nucleic acid of a helper virus, whereby thenucleic acid of the helper virus comprises a nucleic acid sequence whichcodes for the oncogene protein, particularly the E1A protein, which hasthe following characteristics and confers the following characteristicsto the adenovirus, respectively: It is preferably non-replicating inYB-1 nucleus-negative cells but is replicating in cells which areindependent of the cell cycle in YB-1 nucleus-positive or in cellsexhibiting deregulated YB-1, is transactivating at least one viral gene,in particular E1B55 kDa, E4orf6, E4orf3 and/or E3ADP, in YB-1nucleus-positive cells, and/or does not transfer cellular YB-1 into thenucleus. It is within the present invention that the transgenesdescribed herein are either individually or collectively coded and/orexpressed by the helper virus. This applies to helper viruses for bothgroup I adenoviruses and group II adenoviruses.

Furthermore, in an embodiment of such an adenoviral replication systemin accordance with the invention the adenoviral nucleic acid and/or thenucleic acid of the helper virus is/are present as replicable vector.

It is further within the present invention that the nucleic acid(s)coding for group I adenoviruses and/or group II adenoviruses is/arepreferably present in an expression vector and that this expressionvector is used in accordance with the invention.

In a further aspect the present invention is also related to a vectorgroup comprising at least two vectors, whereby the vector groupcomprises in total an adenoviral replication system for group Iadenoviruses and/or group II adenoviruses as described herein, and thevector group is used in accordance with the invention. In an embodimenteach component of the adenoviral replication system is arranged on anindividual vector, preferably an expression vector.

Finally, the present invention is related in a further aspect to the useof a cell which contains one or several of the nucleic acids which codefor the group I adenoviruses and/or group II adenoviruses which arepreferably used in accordance with the present invention, and which areto be used in accordance with the invention of and/or a correspondingadenoviral replication system and/or a corresponding vector and/or avector group according to the invention, for the very same purpose asdescribed herein for the various adenoviruses.

The above described constructs of adenoviruses and in particular theirnucleic acids and the nucleic acids coding therefor, may also beintroduced in a multipartite form into a cell, preferably a tumor cell,whereby due to the presence of the various individual components theyact together as if the individual components were derived from a singlenucleic acid and a single or several adenoviruses, respectively.

The nucleic acids which are used in accordance with the invention andwhich code for group I adenoviruses and/or group II adenoviruses,corresponding adenoviral systems or parts thereof, may also be presentas vectors. Preferably these vectors are viral vectors. In case thenucleic acids comprise adenoviral nucleic acids, preferably the virusparticle is the vector. It is, however, also within the presentinvention that said nucleic acids are present in a plasmid vector. Ineach case the vector comprises elements which allow for and control thepropagation of inserted nucleic acid, i.e. replication and the optionalexpression of the inserted nucleic acid. Suitable vectors, preferablyexpression vectors, and respective elements are known to the onesskilled in the art and, for example, described in Grunhaus, A., Horwitz,M. S., 1994, Adenoviruses as cloning vectors. In Rice, C., editor,Seminars in Virology, London: Saunders Scientific Publications.

The aspect related to the vector groups takes into account theafore-described embodiment that the various elements of said nucleicacid are not necessarily contained in a single vector only. Accordingly,a vector group consists of at least two vectors. Apart from that, anystatements made in relation to the vectors is also applicable to thevectors and the vector group, respectively.

Group I adenoviruses and/or group II adenoviruses are characterised bythe various nucleic acids and gene products, respectively, disclosedherein and may otherwise comprise all those elements known to the onesskilled in the art and which are inherent to the wildtype adenoviruses(Shenk, T.: Adenoviridae: The virus and their replication. FieldsVirology, vol. 3, editors Fields, B. N., Knipe, D. M., Howley, P. M. etal., Lippincott-Raven Publishers, Philadelphia, 1996, chapter 67).

For purpose of illustration but not for purpose of limitation of thepresent invention the replication of adenoviruses shall be brieflydiscussed in the following.

The replication of adenoviruses is a very complex process and is usuallybased on the human transcription factor E2F. During viral infection atfirst the “early genes” E1, E2, E3 and E4 are expressed. The group ofthe “late genes” is responsible for the synthesis of the structuralproteins of the virus. The E1 region consisting of two transcriptionalunits E1A and E1B which code for different E1A and E1B proteins, play acritical role for the activation of both the early and the late genes,as they induce the transcription of the E2, E3 and E4 genes (Nevins, J.R., Cell 26, 213-220, 1981). Additionally, the E1A proteins may initiateDNA synthesis in resting cells and thus trigger their entry into the Sphase (c. f. Boulanger and Blair, 1991). Additionally, they interactwith the tumor suppressors of the Rb class (Whyte, P. et al., Nature334, 124-127, 1988). In doing so, the cellular transcription factor E2Fis released. The E2F factors may subsequently bind to correspondingpromoter regions of both cellular and viral genes (in particular to theadenoviral E2 early promoter) and initiate transcription and thusreplication (Nevins, J. R., Science 258, 424-429, 1992). The activity ofpRb and E2F is regulated by phosphorylation. The hypophosphorylated formof pRb particularly exists in the G1 and M phase. In contrast thereto,the hyperphosphorylated form of pRb is present in the S and G2 phase. Byphosphorylation of pRb E2F is released from the complex consisting ofE2F and hypophosphorylated pRb. The release of E2F from the complex ofE2F and hypophosphorylated pRb results in transcription of E2F dependentgenes. The E1A protein binds only to the hypophosphorylated form of pRb,whereby the binding of E1A to pRb predominantly occurs through the CR2region of the E1A protein. Additionally, it also binds to the CR1region, however, with a lower affinity (Ben-Israel and Kleiberger,Frontiers in Bioscience, 7, 1369-1395, 2002; Helt and Galloway,Carcinogenesis, 24, 159-169, 2003).

The gene products of the E2 region are especially needed for theinitiation and completion of the replication as they code for threeessential proteins. The transcription of the E2 proteins is controlledby two promoters, the “E2 early E2F dependent” promoter, which is alsoreferred to herein as E2-early promoter or early E2 promoter, and the“E2-late” promoter (Swaminathan and Thimmapaya, The Molecular Repertoireof Adenoviruses III: Current Topics in Microbiology and Immunology, vol199, 177-194, Springer Verlag 1995). Additionally, the products of theE4 region together with the E1A and E1B-55 kDa protein play a crucialrole for the activity of E2F and the stability of p53. For example, theE2 promoter is even more transactivated by direct interaction of theE4orf6/7 protein encoded by the E4 region with the heterodimerconsisting of E2F and DP1 (Swaminathan and Thimmapaya, JBC 258, 736-746,1996). Furthermore, the complex consisting of E1B-55 kDa and E4orf6 isinactivated by p53 (Steegenga, W. T. et al., Oncogene 16, 349-357, 1998)in order to complete a successful lytic infectious cycle. Additionally,E1B-55 kDa has a further important function insofar as it promotes, wheninteracting with E4orf6 protein, the export of viral RNA from thenucleus, whereas cellular RNAs are retained in the nucleus (Bridge andKetner, Virology 174, 345-353, 1990). A further important observation isthat the protein complex consisting of E1B-55 kDa/E4orf6 is localised inthe so-called “viral inclusion bodies”. It is assumed that thesestructures are the sites of replication and transcription (Ornelles andShenk, J. Virology 65, 424-429, 1991).

The E3 region is another important region for the replication and inparticular for the release of adenoviruses. The E3 region more preciselycontains the genetic information for a variety of comparatively smallproteins which are not essential for the infectious cycle of adenovirusin vitro, i.e. in cell culture. However, they play a crucial role in thesurvival of the virus during an acute and/or latent infection in vivo asthey have, among others, immune regulatory and apoptotic function(s)(Marshall S. Horwitz, Virologie, 279, 1-8, 2001; Russell, supra). Itcould be shown that a protein having a size of about 11.6 kDa inducescell death. This protein was, due to its function, named ADP—for theenglish term adenovirus death protein—(Tollefson, J. Virology, 70,2296-2306, 1996). The protein is predominantly formed in the late phaseof the infectious cycle. Furthermore, the overexpression of the proteinresults in a better lysis of the infected cells (Doronin et al., J.Virology, 74, 6147-6155, 2000).

Furthermore, it is known to the present inventor that E1A-deletedviruses, i.e. particularly those viruses which neither express any 12SE1A protein nor any 13S E1A protein, may replicate very efficiently athigher MOIs (Nevins J. R., Cell 26, 213-220, 1981), which, however,cannot be realised in clinical applications. This phenomenon is referredto as “E1A-like activity” in literature. Furthermore it was known thatof the 5 proteins encoded by E1A, two proteins, namely the 12S and the13S protein, control and induce, respectively, the expression of theother adenoviral genes (Nevins, J. R., Cell 26, 213-220, 1981;Boulanger, P. and Blair, E.; Biochem. J. 275, 281-299, 1991). It becameevident that particularly the CR3 region of the 13S protein isexhibiting the transactivating function (Wong H K and Ziff E B., JVirol., 68, 4910-20, 1994). Adenoviruses having distinct deletions inthe CR1 and/or CR2 region and/or CR3 region of the 13 S protein areessentially replication-defective, however are still transactivating inother cell lines the viral genes and promoters, and in particular the E2region (Wong H K, Ziff E B., J Virol. 68, 4910-20, 1994; Mymryk, J. S.and Bayley, S. T., Virus Research 33, 89-97, 1994).

After infection of a cell, typically a tumor cell, with a wildtypeadenovirus, YB-1 is induced into the nucleus by means of E1A, E1B-55Kand E4orf6 and co-localised with E1B-55K in the viral inclusion bodieswithin the nucleus which allows an effective replication of the virus inthe cellular nucleus both in vitro and in vivo. It has been foundalready earlier that E4orf6 also binds to E1B-55K (Weigel, s. andDobbelstein, M. J. Virology, 74, 764-772, 2000; Keith N. Leppard,Seminars in Virology, 8, 301-307, 1998) and thus mediates the transportand distribution of E1B-55K into the nucleus which ensures an optimumvirus production and adenoviral replication, respectively. By theco-operation of E1A, E1B-55K and YB-1, and by the complex consisting ofE1B-55K/E4orf6 and YB-1, respectively, and the co-localisation of YB-1and E1B-55K in the nucleus in the so-called viral inclusion bodies, anefficient replication of the virus in accordance with the invention ispossible and thus the use of the viruses described herein forreplication in cells which are YB-1 nucleus-positive, preferably cellswhich contain YB-1 in the nucleus independent of the cell cycle, and/orcells which comprise or exhibit deregulated YB-1, and/or for themanufacture of a medicament, respectively, for the treatment ofdiseases, in which YB-1 nucleus-positive cells, preferably cells whichcontain YB-1 in the nucleus independent of the cell cycle, and/or cellswhich comprise or exhibit deregulated YB-1, are involved. Thereplication which is therefore possible in this cellular background,results in lysis of the cell, release of the virus and infection andlysis of adjacent cells so that in case of infection of a tumor cell anda tumor, respectively, finally lysis of the tumor, i.e. oncolysis,occurs.

YB-1 belongs to a group of highly conserved factors which bind to aninverted CAAT sequence which is referred to as Y-box. They may act in aregulatory manner both at the level of transcription and translation(Wolffe, A. P. Trends in Cell Biology 8, 318-323, 1998). There are moreand more Y-box dependent regulation pathways found in the activation butalso in the inhibition of growth and apoptosis associated genes(Swamynathan, S. K. et al., FASEB J. 12, 515-522, 1998). For example,YB-1 interacts directly with p53 (Okamoto, T. et al., Oncogene 19,6194-6202, 2000), plays an essential role in the expression of the Fasgene (Lasham, A. et al., Gene 252, 1-13, 2000), in gene expression ofMDR and MRP (Stein, U. et al., JBC 276, 28562-69, 2001; Bargou, R. C. etal., Nature Medicine 3, 447-450, 1997) and in the activation oftopoisomerases and metalloproteinases (Mertens, P. R. et al., JBC 272,22905-22912, 1997; Shibao, K. et al., Int. J. Cancer 83, 732-737, 1999).Also, YB-1 is involved in the regulation of mRNA stability (Chen, C-Y.et al., Genes & Development 14, 1236-1248, 2000) and in repair processes(Ohga, T. et al., Cancer Res. 56, 4224-4228, 1996; Izumi H. et al.,Nucleic Acid Research 2001, 29, 1200-1207; Ise T. et al., Cancer Res.,1999, 59, 342-346).

The nuclear localisation of YB-1 in tumor cells either by YB-1 beingpresent in the nucleus independent of the cell cycle, or by deregulatedYB-1 present in the cytoplasm having been translocated into the nucleusby group I adenoviruses and/or group II adenoviruses, results inE1A-independent viral replication during which especially neither any12S E1A protein nor any 13S E1A protein is expressed and used,respectively (Holm, P. S. et al. JBC 277, 10427-10434, 2002), andresults in a multidrug resistance in case of overexpression of theprotein YB-1. Additionally, it is known that the adenoviral proteinssuch as, e.g., E4orf6 and E1B-55K have a positive impact on viralreplication (Goodrum, F. D. and Ornelles, D. A., J. Virology 73,7474-7488, 1999), whereby a functional E1A protein is responsible forthe activation of the other viral gene products (such as E4orf6, E3ADPand E1B-55K) (Nevins J. R., Cell 26, 213-220, 1981). This, however, doesnot happen with the E1A-minus adenoviruses known in the art in which the13S E1A protein is not present. Nuclear localisation of YB-1 inmultidrug resistant cells which have YB-1 in the nucleus, allows for thereplication and particle formation, respectively, of such E1A-minusviruses. In connection therewith, however, the efficiency of viralreplication and particle formation is reduced compared to the wildtypeAd5 by a multiple. Compared to this, a combination of YB-1 allows for avery efficient viral replication and particle formation mediated by YB-1and thus oncolysis, whereby the YB-1 is either already contained in thenucleus of the tumor cell which may result from YB-1 being located inthe nucleus in a cell cycle independent manner, or whereby thederegulated YB-1 present in the cytoplasm is translocated into thenucleus by group I adenoviruses and/or group II adenoviruses, or isinduced into the cellular nucleus by exogenous factors (e.g. applicationof cytostatics or irradiation or hyperthermia), i.e. is induced to bepresent in the nucleus, particularly independent of the cell cycle, orwhereby YB-1 is introduced as a transgene by a vector with a system,preferably an adenoviral system, which switches on the adenoviral genesbut does not show viral replication. This applies also to theadenoviruses in accordance with the invention, i.e. group Iadenoviruses, which are capable of efficiently replicating due to theirspecific design and using the effect that an E1B protein, preferably theE1B55K protein, and/or an E4 protein, preferably the E4orf6 protein,provide(s) for an effective mobilisation of YB-1, preferably in thenucleus. Suitable cytostatics which may be used together with theadenoviruses disclosed herein in connection with the various aspects ofthe present invention are, for example, those belonging to the followinggroups: anthracyclines such as for example daunomycin and adriamycin;alkylating agents such as for example cyclophosphamide; alkaloides suchas etoposide; vin-alkaloides such as for example vincristine andvinblastine; antimetabolites such as for example 5-fluorouracil andmethrothrexat; platin-derivatives such as for example cis-platin;topoisomerase inhibitors such as for example camphothecine, CPT-11;taxanes such as for example taxole, paclitaxel, histone-deacetylaseinhibitors such as for example FR901228, MS-27-275, trichostatine A, MDRmodulators such as for example MS-209, VX-710 and geldanamycinederivatives such as for example 17-AAG. The adenoviruses disclosedherein, in particular recombinant adenoviruses, which are only capableof replicating in cells which are YB-1 nucleus-positive, and cells whichcontain regulated YB-1, preferably in the cytoplasm, are limited intheir ability to transactivate the viral genes E1B-55K, E4orf6, E4orf3and E3ADP compared to the respective transactivating abilities ofwildtype adenoviruses, in particular wildtype Ad5. The present inventorhas surprisingly found that this limited transactivating ability can beovercome by expressing the corresponding genes, and in particularE1B-55K and E4orf6, in combination with the nuclear localisation ofYB-1. As shown in the examples herein, viral replication and particleformation is increased under such conditions to a level comparable tothe replication activity and particle formation activity, respectively,of wildtype adenoviruses.

The medicament in connection with which or for the manufacture of whichthe adenoviruses disclosed herein are used in accordance with thepresent invention, is intended to be applied, usually, in a systemicmanner, although it is also within the present invention to apply ordeliver it locally. The application is intended to infect particularlythose cells with adenoviruses and it is intended that adenoviralreplication particularly occurs therein, which are involved, preferablyin a causal manner, in the formation of a condition, typically adisease, for the diagnosis and/or prevention and/or treatment of whichthe inventive medicament is used.

Such a medicament is preferably for the treatment of malignant diseases,tumor diseases, cancer diseases, cancer and tumors, whereby these termsare used herein in an essentially synonymous manner if not indicated tothe contrary. The tumor diseases are preferably those where YB-1 is, dueto the mechanism underlying the tumor disease, in particular due to theunderlying pathological mechanism, already located in the nucleus,preferably independent of the cell cycle, or where the presence of YB-1in the cellular nucleus is caused by exogenous measures whereby suchexogenous measures are suitable to transfer YB-1 into the cellularnucleus or to induce or to express it there. The term tumor or tumordisease shall comprise herein both malignant as well as benign tumors,each both solid and diffuse tumors, and respective diseases. In anembodiment the medicament comprises at least one furtherpharmaceutically active compound. The nature and the amount of suchfurther pharmaceutically active compound will depend on the kind ofindication for which the medicament is used. In case the medicament isused for the treatment and/or prevention of tumor diseases, typicallycytostatics such as cis-platin and taxole, daunoblastin, daunorubicin,adriamycin and/or mitoxantrone or others of the cytostatics or groups ofcytostatics described herein, are used, preferably those as described inconnection with the cytostatic mediated nuclear localisation of YB-1.

The medicament in accordance with the invention can be present invarious formulations, preferably in a liquid form. Furthermore, themedicament will contain adjuvants such as stabilisers, buffers,preservatives and the like which are known to the one skilled in the artof formulations.

The present inventor has furthermore surprisingly found that theefficacy of the viruses described herein and in particular the virusesused in accordance with the present invention can be increased by usingthem in combination with at least two agents whereby each of the atleast two agents is individually and independently selected from thegroup comprising cytostatics.

As used herein in a preferred embodiment, cytostatics are in particularchemical or biological compounds which, during or after theadministration to a cell or an organism containing a or such cell,result in the cell no longer growing and/or no longer dividing orslowing down cell division and/or cell growth. Cytostatics also comprisecompounds which turn into a cytostatic in the afore-described sense onlyin the cell or in an organism containing such cell. Insofar, the termcytostatics also comprises pre-cytostatics.

Cytostatics are grouped according to their mode of action. The followinggroups are distinguished which, in principle, can all be used within thepresent invention:

Alkylating agents, i.e. chemical compounds which cause their cytotoxiceffect by alkylating phosphate, amino, sulphydryl, carboxy and hydroxygroups of the nucleic acid as well as proteins. Such compounds are oftencancerogenic themselves. Typical examples of this group of cytostaticsare cis-platin and platin derivatives, cyclophosphamide, dacarbazine,mitomycin, procarbazine.

Antimetabolites, i.e. compounds which, due to their structuralsimilarity or ability for binding block a metabolic process or affectthe same. Within the group of antimetabolites it is distinguishedbetween structurally similar antimetabolites, structure changingantimetabolites and the indirectly acting antimetabolites. Thestructurally similar antimetabolites compete due to chemical similaritywith the metabolite without exerting the function thereof. Structurechanging antimetabolites bind to the metabolites which impedes itsfunction or resorption or chemically modifies the metabolite. Indirectlyacting antimetabolites interfere with the function of the metabolite,for example by the binding of ions. Typical examples of this group arefolic acid antagonists such as methotrexate, pyrimidine analogues suchas fluorouracil, purine analogues such as azathioprine andmercaptopurine.

Mitosis inhibitors, i.e. compounds which inhibit cell division. Withinthe group of mitosis inhibitors it is distinguished between celldivision toxins, spindle toxins and chromosome toxins. Typical examplesof this group are taxanes and vinca alkaloids. The taxanes in turn canbe divided into the two major groups of taxoles and taxoters, whereby aparticularly preferred taxole is paclitaxel, and a particularlypreferred taxoter is docetaxel.

Antibiotics having an inhibitory effect on the DNA-dependent RNApolymerase. Typical examples are the anthracyclines, such as, e.g.,bleomycin, daunorubicin, doxorubicin and mitomycin.

Topoisomerase inhibitors, in particular topoisomerase I inhibitors.Topoisomerase inhibitors are chemical compounds which determine thetertiary structure of the DNA by catalysing the change of the DNA twistnumber in a three stage process. Essentially, two forms oftopoisomerases are distinguished. Topoisomerases of type I cleave only aDNA strand and are ATP-independent, whereas topoisomerase of type IIcleave both strands of a DNA, whereby they are ATP-dependent. Typicalexamples for topoisomerase I inhibitors are irinotecan and topotecan,and for topoisomersae II inhibitors etoposid and daunorubicin.

Within the present invention at least one and preferably two agents areselected from the aforementioned group. It is, however, also within theinvention that in particular also three, four or five different agentsare selected. The following comments are made for the embodiment of thepresent invention where only two agents are used together with thevirus. These considerations are basically also applicable to embodimentswhere more than two agents are used.

Preferably the agents differ from each other such that they address ortarget different target molecules or are described in the literature astargeting different molecules. It is within the present invention thatthe agent also comprises two or more different agents which bind to thesame target molecule. It is also within the present invention that oneagent binds to a first site of the target molecule, whereas the secondagent binds to a second site of the target molecule.

It is also within the present invention that at least two of the agentsare active using different modes of action. Active means in a preferredembodiment that the cell growth and/or cell division inhibiting orretarding effect of the chemical compound is mediated through adifferent mode of action. In a particularly preferred embodiment theterm active means that the replication efficiency of a virus, inparticular the virus in accordance with the present invention, of theviruses described herein and of the viruses to be used in accordancewith the present invention, is increased compared to a scenario whereone and/or both of the agents are not used. As a measure for theefficiency of viral replication preferably the number of virusesrequired for cell lysis is used, preferably expressed as pfu/cell.

In a particularly preferred embodiment at least one of the at least twoagents is one which increases the infectability of the cell in which thereplication of the virus is to occur, preferably is to occur in aselective manner, preferably with the virus described herein and/or thevirus to be used in accordance with the present invention. This can,e.g., be performed by increasing the uptake of the virus by the cell.The uptake of the virus, in particular of adenovirus, is, for example,mediated by the coxsackievirus-adenovirus receptor (CAR) (Mizuguchi undHayakawa, GENE 285, 69-77, 2002). An increased expression of CAR is, forexample, caused by trichostatin A (Vigushin et al., Clinical CancerResearch, 7, 971-976, 2001).

In a further embodiment one of the at least two agents is one whichincreases the availability of a component within the cell, whereby thecomponent is one which increases the replication of the virus,preferably the virus described herein and/or the virus to be used inaccordance with the present invention.

In a further embodiment one of the at least two agents is one whichmediates the transport of YB-1 into the nucleus. Such an agent can beselected from the group comprising topoisomerase inhibitors, alkylatingagents, antimetabolites and mitosis inhibitors. Preferred topoisomeraseinhibitors are camptothecin, irinotecan, etoposide and their respectiveanalogues. Preferred mitosis inhibitors are daunorubicin, doxorubicin,paclitaxel and docetaxel. Preferred alkylating agents are cis-platin andtheir analogues. Preferred antimetabolites are fluorouracil andmethotrexat.

In a particularly preferred embodiment one of the at least two agents isone which increases the infectability of the cell, in particular theexpression of CAR, and the second of the at least two agents is onewhich increases the transport of YB-1 into the nucleus, wherebypreferably as chemical compound a compound is used which exhibits therespective required characteristic as preferably described above.

In a further embodiment the one of the at least two agents is a histonedeacylase inhibitor. A preferred histone deacylase inhibitor is onewhich is selected from the group comprising trichostatin A, FR901228,MS-27-275, NVP-LAQ824 and PXD101. Trichostatin A is, for example,described in Vigushin et al., Clinical Cancer Research, 7, 971-976,2001; FR901228 is, for example, described in Kitazono et al., CancerRes., 61, 6328-6330, 2001; MS-27-275 is described in Jaboin et al.,Cancer Res., 62, 6108-6115, 2002; PXD11 is described in Plumb et al.,Mol. Cancer Ther., 8, 721-728, 2003; NVP-LAQ824 is described in Atadjaet al., Cancer Res., 64, 689-695, 2004.

In a still further embodiment the one of the at least two agents is atopoisomerase inhibitor, preferably a topoisomerase I inhibitor. Apreferred topoisomerse inhibitor is one which is selected from the groupcomprising camptothecin, irinotecan, topotecan, SN-38,9-aminocamptothecin, 9-nitrocamptothecin, DX-895If and daunorubicin.Irinotecan and SN-38 are, for example, described in Gilbert et al.,Clinical Cancer Res., 9, 2940-2949, 2003; DX-8951F is described in vanHattum et al., British Journal of Cancer, 87, 665-672, 2002;camptothecin is described in Avemann et al., Mol. Cell. Biol., 8,3026-3034, 1988; 9-aminocamptothecin, 9-nitrocamptothecin are describedin Rajendra et al., Cancer Res., 63, 3228-3233, 2003; daunorubicin isdescribed in M. Binaschi et al., Mol. Pharmacol., 51, 1053-1059.

In a particularly preferred embodiment the one of the at least twoagents is a histone deacylase inhibitor and the other one of the atleast two agents is a topoisomerse inhibitor.

In an embodiment the means according to the present invention and/or themeans prepared in accordance with the present invention contains thevirus separate from one or several of the at least two agents which arecombined with the virus in accordance with the present invention. It ispreferred that the virus is separate from any agent which is combinedwith the virus. Preferably the separation is a spatial separation. Thespatial separation can be such that the virus is present in a differentpackage than the agent. Preferably the package is a single dose unit,i.e. the virus and the agent are packed as single dosages or doses. Thesingle dose units may in turn be combined to form a package. However, itis also within the present invention that the single dosages of thevirus are combined with one or several single dosages of one or severalof the agents or are packed therewith.

The kind of package depends on the way of administration as known to theone skilled in the art. Preferably the virus will be present in alyophilized form or in a suitable liquid phase. Preferably, the agentswill be present in solid form, e.g. as tablets or capsules, however, arenot limited thereto. Alternatively, also the agents can be present inliquid form.

It is within the present invention that the virus is systemically orlocally administered. It is also within the present invention that theagents combined with the virus are systemically or locally administeredindividually and independently from each other or together. Other modesof administration are known to the ones skilled in the art.

It is also within the present invention that the virus and the agentscombined with it, are administered in a chronologically separate manneror at the same time. In connection with a chronologically separatemanner it is preferred that the agents are administered prior to theadministration of the virus. How long the agent is administered prior tothe virus depends on the kind of the agent used and is obvious for theone skilled in the art from the mode of action of the agent used. Alsothe administration of the at least two agents can occur at the same orat different points in time. In connection with a chronologicallydifferent administration the points of time again result from the modesof action underlying the agents and can, based thereon, be determined bythe ones skilled in the art.

The above considerations, given in connection with the medicamentsaccording to the present invention which are also disclosed and referredto herein as pharmaceutical compositions, are roughly also applicable toany composition, including compositions as used for the replication ofviruses, preferably for the in vitro replication of viruses inaccordance with the present invention. The above considerations are alsoapplicable to the kit in accordance with the present invention and thekit to be used in accordance with the present invention, respectively,which may apart from the viruses described herein and the viruses to beused in accordance with the invention, also comprise one agent or acombination of agents as described herein. Such kits comprise the virusand/or one or several agents in a form ready for use, and preferablyinstructions for use. Furthermore, the above embodiments apply also tothe nucleic acids as disclosed herein, and the nucleic acids used inaccordance with the present invention, and the replication systems inaccordance with the present invention and the nucleic acids codingtherefor, and, respectivel, the replication systems used in accordancewith the present invention and the nucleic acids coding therefor used inaccordance with the present invention.

The present inventor has surprisingly found that the inventive use ofthe viruses described herein, preferably the use of group I adenovirusesand/or group II adenoviruses can be practised with a very high rate ofsuccess in connection with such tumors and that they can be used for themanufacture of medicaments for the treatment of such tumors, which haveYB-1 in the cellular nucleus independent of the cell cycle. Normally,YB-1 is located in the cytoplasm, in particular in the perinuclearplasm. In the G1/S phase of the cell cycle YB-1 can be found in thenucleus of both normal as well as tumor cells, whereby part of the YB-1remains in the cytoplasm [Jürchott K et al., JBC 2003, 278,27988-27996]. This, however, is not sufficient in order to provide forviral oncolysis using such modified adenoviruses. The comparatively lowefficacy of such attenuated adenoviruses as described in the prior art,is ultimately based on their wrong application. In other words, suchadenoviral systems may be particularly used with a higher efficiency incase where the molecular biological prerequisites for viral oncolysis isgiven using these attenuated or modified adenoviruses as describedherein, preferably using the group I adenoviruses and/or group IIadenoviruses. In case of the adenoviruses described herein to be used inaccordance with the present invention, such as AdΔ24, dl922-947,E1Ad/01/07, CB016, dl520 and the recombinant adenoviruses described inEuropean patent EP 0 931 830, the prerequisites are given in such tumorsthe cells of which show a cell cycle independent nuclear localisation ofYB-1. This kind of nuclear localisation may be caused by the nature ofthe tumor itself or by the measures or inventive agents according to theinvention as described herein. The present invention thus defines a newgroup of tumors and tumor diseases and thus also of patients which maystill be efficiently treated using the viruses according to theinvention as, preferably group I adenoviruses and/or group II viruses,but also using attenuated or modified adenoviruses already described inthe prior art.

A further group of patients which may be treated in accordance with theinvention using group I adenoviruses and/or group II adenoviruses or theadenoviruses to be used in accordance with the present invention whichare, as such, already known in the prior art, or using the adenovirusesdescribed herein for the very first time, and preferably using suchadenoviruses which have a mutation and deletion, respectively, in theE1A protein which does not interfere with the binding of Rb/E2f or whichare not replicating in YB-1 nucleus-negative cells or which show astrongly reduced replication as defined herein, and/or which have and/orshow a deleted oncoprotein, in particular E1A, such as, for example, incase of the viruses AdΔ24, dl922-947, E1Ad/01/07, CB106 and theadenoviruses described in European patent EP 0 931 830, are thosepatients in which it is ensured that by applying or realising specificconditions that YB-1 migrates into the nucleus or is induced ortransported there, or that deregulated YB-1 is present. The use of groupI adenoviruses and/or group II adenoviruses in connection with thisgroup of patients is based on the finding that the induction of viralreplication is based on nuclear localisation of YB-1 with subsequentbinding of YB-1 to the E2-late promoter. Because of this findingdisclosed herein, adenoviruses such as AdΔ24, dl922-947, E1Ad/01/07,CB106 and/or the adenoviruses described in European patent EP 0 931 830may also replicate in such cells which are YB-1 nucleus-positive and/orin cells in which YB-1 is present in a deregulated manner in the meaningof the present invention. Insofar these adenoviruses can be used for thetreatment of diseases and patient groups in accordance with the presentinvention which/who comprise cells having these characteristics,particularly if these cells are involved in the generation of therespective disease to be treated. This is the basis for the success ofAdΔ24, dl922-947, E1Ad/01/07, CB106, of the adenoviruses described inpatent EP 0 931 830 and of the group I adenoviruses and/or group IIadenoviruses in the treatment according to the invention of such tumorswhich contain YB-1 in the nucleus independent of the cell cycle or whichcontain deregulated YB-1 in the meaning of the present disclosure. Afurther group of patients which may be treated in accordance with thepresent invention using the viruses described herein as to be used inaccordance with the present invention and using the viruses describedherein for the very first time, particularly adenoviruses and group Iand/or group II adenoviruses, respectively, are those which are YB-1nucleus-positive and/or YB-1 nucleus-positive as a result of any of thetreatments in the following, and/or such patients which will undergo oneof the following measures, preferably in the sense of a treatment, priorto the administration of the adenoviruses, concomitant with theapplication of the respective viruses or after the administration ofadenoviruses. It is within the present invention that YB-1nucleus-positive patients are patients who in particular have YB-1 inthe nucleus in a number of the tumor forming cells independent of thecell cycle, and/or have deregulated YB-1 in such cells. One of thesemeasures is the administration of cytostatics as described herein as awhole and/or as used in connection with tumor therapy. Furthermore,irradiation belongs to this group of measures, particularly irradiationas applied in the tumor therapy. Irradiation particularly means theirradiation with high energy radiation, preferably radioactiveradiation, preferably as used in tumor therapy. A further measure ishyperthermia and the application of hyperthermia, respectively,preferably hyperthermia as used in tumor therapy. In a particularlypreferred embodiment hyperthermia is applied locally. Finally, a furthermeasure is hormone treatment, particularly hormone treatment as appliedin tumor therapy. In the course of such hormone therapy anti-estrogensand/or anti-androgens are used. In connection therewith, anti-estrogenssuch as tamoxifen, are particularly used in the treatment of breastcancer, and anti-endrogens, such as for example flutamide orcyproteronacetate, are particularly used in the therapy of prostatecancer.

The adenoviruses disclosed herein may also be used for the treatment oftumors, whereby the tumor is selected from the group comprising primarytumors, secondary tumors, tertiary tumors and metastatic tumors. Inconnection therewith it is preferred that the tumors exhibit at leastone of the following features, namely that they have YB-1 in the nucleusindependent of the cell cycle, irrespective of what the reason for thisis, and/or that they contain deregulated YB-1.

It is within the present invention that the cells and the tumors,respectively, comprising such cells in which the adenoviruses inaccordance with the invention replicate or are capable of replicating,are those which have one or several of the features described herein,particularly the feature that they have YB-1 in the nucleus independentof the cell cycle, regardless of the reason therefor, and/or the featurethat they have deregulated YB-1, and that these cells and tumors,respectively, may be treated using the group I adenoviruses and/or groupII adenoviruses in accordance with the present invention, and that theadenoviruses may be used for the manufacture of a medicament for thetreatment of them, whereby the adenoviruses express a YB-1 codingnucleic acid. Therefore, there are preferably three categories of cellsand thus of tumors in which the group I adenoviruses and group IIadenoviruses in accordance with the present invention may replicate andwhich may be treated and preferably lysed using these adenoviruses,respectively:

-   -   Group A: Cells which have YB-1 in the nucleus independent of the        cell cycle;    -   Group B: Cells which do not have YB-1 in the nucleus,        particularly not independent of the cell cycle, but comprise        deregulated YB-1; and    -   Group C: Cells which do not have YB-1 in the nucleus,        particularly not independent of the cell cycle, and which do not        comprise deregulated YB-1.

For the cells of group A the adenoviruses in accordance with the presentinvention, particularly group I adenoviruses, which do not expressadditional YB-1, may be used for replication or lysis. However, it isalso possible that such adenoviruses in accordance with the presentinvention, in particular group I adenoviruses which express additionalYB-1, are used for replication and lysis. This applies also to group B.Without wishing to be bound thereto, the reason seems to be that due tothe effect of the E1B protein, in particular the E1B55K protein, and/orthe E4 protein, particularly the E4orf6 protein, an efficientreplication is ensured by localisation of YB-1 in the nucleus and thetransfer of the same to the nucleus, respectively. YB-1 additionallyexpressed by the adenoviruses, supports this process.

In case of group C, preferably those adenoviruses in accordance with theinvention, particularly group I adenoviruses will be used forreplication or lysis which additionally express YB-1. The reason forthis seems to be, again without wishing to be bound thereto, that theabove processes of viral replication are not active in the particularcellular background such that an efficient replication may occur. Onlyby providing YB-1 and expressing YB-1, respectively, an efficientreplication may occur, whereby the underlying mechanism seems to be suchthat the overexpression of YB-1 results in nuclear localisation of YB-1as also described by Bargou [Bargout R. C. et al., Nature Medicine 1997,3, 447-450) and Jürchott [Jürchott K. et al., JBC 2003, 278,27988-27966].

It is within the present invention that some of the tumor forming cellswhich either inherently contain YB-1 in the nucleus or do so or afterinduction and active introduction into the nucleus or which comprisederegulated YB-1 in the meaning of the present disclosure. Preferablyabout 5% or any percentage higher than that, i.e. 6%, 7%, 8% etc., ofthe tumor forming cells are such YB-1 nucleus-positive cells or cells inwhich deregulated YB-1 is present. For other tumors such as breasttumor, osteosarcoma, ovarian carcinoma, synovial carcinoma or lungcarcinoma the percentage of tumor cells which comprise deregulated YB-1or which show nuclear localisation of YB-1 independent of the cellcycle, may be about 30 to 50% [Kohno K. et al., BioEssays 2003, 25,691-698]. Such tumors may preferably be treated using the adenovirusesin accordance with the present invention. Nuclear localisation of YB-1may be induced by outside stress and locally applied stress,respectively. This induction may occur through irradiation, particularlyUV-irradiation, application of cytostatics as, among others, alsodisclosed herein, and hyperthermia. In connection with hyperthermia itis important that it may be realized in a very specific manner,particularly a local manner, and that thus also a specific nucleartransport of YB-1 into the nucleus may be caused and, because of this,the prerequisites for replication of the adenovirus and thus of cell andtumor lysis are given, which preferably is locally limited (Stein U,Jurchott K, Walther W, Bergmann, S, Schlag P M, Royer H D. J Biol Chem.2001, 276(30):28562-9; Hu Z, Jin S, Scotto K W. J Biol Chem. 2000 Jan.28; 275(4):2979-85; Ohga T, Uchiumi T, Makino Y, Koike K, Wada M, KuwanoM, Kohno K. J Biol Chem. 1998, 273(11):5997-6000).

The medicament of the invention would thus also be administered topatients and groups of patients or would be designed for them, where byappropriate pre- or post-treatment or concomitant treatment a transportof YB-1, particularly in the respective tumor cells, is caused andderegulated YB-1 is generated in the cell, respectively.

Based on the technical teaching provided herein, it is within the skillsof the one of the art to suitably modify particularly E1A which may, forexample, comprise the generation of deletions or point mutations inorder to thus generate different embodiments of the adenoviruses whichmay be applied in the use in accordance with the present invention.

As already mentioned, group I and/or group II adenoviruses are capableof replicating in such cells and cellular systems, which have YB-1 inthe nucleus. For the question whether also these adenoviruses used inaccordance with the invention are capable of replicating and are thuscapable of tumor lysis, the status of the cells with regard to thepresence or absence of Rb, i.e. the retinoblastome tumor suppressorproduct, is irrelevant. Furthermore, for the use of said adenoviruses inaccordance with the present invention, it is not necessary to take intoaccount the p53 status of the infected cells, of the cells to beinfected or of the cells to be treated as, when using the adenoviralsystems disclosed herein in connection with YB-1 nucleus-positive cells,i.e. cells having YB-1 in the nucleus independent of the cell status,the p53 status as well as the Rb status does not have any impact on thereplication of the adenovirus for the practising the technical teachingdisclosed herein.

The transactivating oncogene and oncogene protein, respectively, inparticular E1A, preferably of the group II adenoviruses, can be eitherunder the control of the proprietary natural adenoviral promoters and/orbe controlled through a tumor-specific or tissue-specific promoter.Suitable non-adenoviral promoters can be selected from the groupcomprising cytomegalovirus promoter, RSV (rous sarcoma virus) promoter,adenovirus-based promoter Va I and the non-viral YB-1 promoter (MakinoY. et al., Nucleic Acids Res. 1996, 15, 1873-1878). Further promoterswhich may be used in connection with any aspect of the inventiondisclosed herein, are the telomerase promoter, the alpha-fetoprotein(AFP) promoter, the caecinoembryonic antigen promoter (CEA) (Cao, G.,Kuriyama, S., Gao, J., Mitoro, A., Cui, L., Nakatani, T., Zhang, X.,Kikukawa, M., Pan, X., Fukui, H., Qi, Z. Int. J. Cancer, 78, 242-247,1998), the L-plastin promoter (Chung, I., Schwartz, P E., Crystal, R C.,Pizzomo, G, Leavitt, J., Deisseroth, A B. Cancer Gene Therapy, 6,99-106, 1999), argenine vasopressin promoter (Coulson, J M, Staley, J.,Woll, P J. British J. Cancer, 80, 1935-1944, 1999), E2f promoter(Tsukada et al., Cancer Res., 62, 3428-3477, 2002), uroplakin IIpromoter (Zhang et al., Cancer Res., 62, 3743-3750, 2002) and the PSApromoter (Hallenbeck P L, Chang, Y N, Hay, C, Golightly, D., Stewart,D., Lin, J., Phipps, S., Chiang, Y L. Human Gene Therapy, 10, 1721-1733,1999), tyrosinase promoter (Nettelbeck, DM. Anti-Cancer Drugs, 14,577-584, 2003), cyclooxygenase 2 promoter (Nettelbeck, D M., Rivera, AA, Davydova, J., Dieckmann, D., Yamamoto, M., Curiel, D T. MelanomaRes., 13, 287-292, 2003) and inducing systems such as tetracycline (Xu,X L., Mizuguchi, H., Mayumi, T., Hayakawa, T. Gene, 309, 145-151, 2003).Furthermore, the YB-1 dependent E2 late promoter of adenoviruses asdescribed in German patent application DE 101 50 984.7 is a promoterwhich may be used in connection with the present invention.

About the telomerase promoter it is known that it is of crucialimportance in human cells.

Accordingly, telomerase activity is regulated through transcriptionalcontrol of the telomerase reverse transcriptase gene (hTERT) which is acatalytic subunit of the enzyme. Expression of the telomerase is activein 85% of the human tumor cells. In contrast thereto it is inactive inmost normal cells. Except therefrom are germ cells and embryonic tissue(Braunstein, I. et al., Cancer Research, 61, 5529-5536, 2001; Majumdar,A. S. et al., Gene Therapy 8, 568-578, 2001). Detailed studies of thehTERT promoter have shown that the fragments of the promoter separatedfrom the initiation codon 283 bp and 82 bp, respectively, are sufficientfor specific expression in tumor cells (Braunstein I. et al.; Majumdar AS et al., supra). Therefore, this promoter and the specific fragments,respectively, are suitable for specific expression in tumor cells of agene and in particular of a transgene, preferably one of the transgenesdisclosed herein. The promoter is to allow for the expression of themodified oncogene, preferably the E1A oncogene protein, in tumor cellsonly. Also, in an embodiment the expression of a transgene in anadenoviral vector under the control of these promoters is contemplated,preferably of a transgene which is selected from the group comprisingE4orf6, E1B55 kD, ADP and YB-1. It is also within the present inventionthat the reading frame of the transactivating oncogene protein, inparticular the E1A protein is in frame with one or several gene productsof the adenoviral system. The reading frame of the transactivating E1Aprotein, however, may also be independent therefrom.

The various transgenes, thus also E1B55 kD, E4orf6, ADP and the like, inparticular if they are viral genes, can, in principle, be cloned fromany respective virus, preferably adenovirus. In the prior artfurthermore a multitude of plasmids is described which containrespective genes and from which these may subsequently be taken andintroduced into the adenoviruses of the present invention as well asinto the viruses used in accordance with the present invention. Anexample for such a plasmid which expresses E1B55 kD, is, for example,described by Dobbelstein, M. et al., EMBO Journal, 16, 4276-4284, 1997.The coding region of the E1B55K gene can be excised together with the 3′non-translating region for example from this gene by Bam HI from plasmidpDCRE1B. The corresponding fragment comprising the E1B55 kD gene as wellas the 3′ non-coding region corresponds to nucleotides 20194107 ofadenovirus type 5. It is, however, also within the present inventionthat the E1B55 kD gene is excised by means of restriction enzyme Bam HIand BfrI from said plasmid and is subsequently cloned into theadenovirus.

It is within the present invention that, if not indicated to thecontrary, in case it is referred to viruses of the present invention itis referred to both the nucleic acid coding therefore as well as theadenoviral particles, preferably including the respective nucleic acid.The respective nucleic acid may also be present as being integrated in adifferent vector.

It is within the present invention that the various promoters describedabove are also used in connection with the various embodiments of theadenoviruses in accordance with the invention, preferably the group Iadenoviruses, particularly in case a promoter is to be used which isdifferent from the one which controls the expression of the respectiveprotein or expression product in wildtype adenoviruses. Theaforementioned promoters are thus suitable heterologous promoters in themeaning of the present invention. In preferred embodiments of theadenoviruses in accordance with the invention, particularly the group Iadenoviruses, it is contemplated that when applying the adenoviruses forcells of group A and B as defined above, this occurs such that theexpression of the E1B protein and/or the E4 protein starts from suchheterologous promoters, whereby preferably, but not exclusively, theexpression of the E1A protein is controlled by YB-1. The expression ofthe E1A protein is in this and other embodiments under the control of aYB-1 controllable promoter such as for example the adenoviral E2-latepromoter. This is also true in that case where the E1B protein and/orthe E4 protein is/are expressed in an expression cassette.

In preferred embodiments of the adenoviruses in accordance with theinvention, particularly the group I adenoviruses, it is contemplatedthat when applying the adenoviruses in connection with cells of group Cthe promoter is each and independently a tumor-specific, organ-specificor tissue-specific promoter. In connection therewith it is sufficientwhen at least one of the promoters which control the expression of theE1B protein, the E4 protein and/or the E1A protein, is such a specificpromoter. By this tumor, organ and tissue specificity, it is ensuredthat replication of the adenoviruses in accordance with the inventionhappens only in cells of the respective tumor, organ or tissue and that,apart from that, no further tissue is damaged by the replication of theadenoviruses such as, for example, is lysed. Preferably, still a secondand more preferably all three proteins are controlled by suchtumor-specific, organ-specific or tissue-specific promoters. Using suchadenoviruses it is possible to lyse also those cells which do not form atumor or which cannot develop into such tumor, but which are for otherreasons such as medicinal reasons to be destroyed or to be removed fromthe organism, preferably a mammalian and more preferably a humanorganism, for example because they produce an undesired factor orproduce such factor at a too high level.

It is contemplated that, in an embodiment, the cells for the lysis ofwhich the described adenoviruses in accordance with the invention areused, are resistant, preferably show a multiple resistance.

Resistances as referred to herein and which are characteristic for thetumors and patients to be treated, are those which are mediated by thefollowing genes, however, are not limited thereto: MDR, MRP,topoisomerase, BCL2, glutathione-2-transferase (GST), protein kinase C(PKC). As the effect of cytostatics is based, among others, on theinduction of apoptosis, the expression of apoptosis-relevant genes playsa crucial role in the generation of any resistance so that the followingfactors are also relevant with regard thereto, namely Fas, the BCL2family, HSP 70 and EGFR [Kim et al., Cancer Chemther. Pharmacol. 2002,50, 343-352].

It has been described by Levenson et al. [Levenson, V. V. et al., CancerRes., 2000, 60, 5027-5030] that the expression of YB-1 is stronglyincreased in resistant tumor cells compared to non-resistant tumorcells.

Resistance as used herein, preferably refers to a resistance against thecytostatics described herein. This multiple resistance preferably goesalong with the expression, preferably an overexpression, of the membranebound transporter protein P glycoprotein which may be used as a markerfor determining respective cells and thus also of tumors exhibiting suchmarker and respective patient groups. The term resistance as used hereinalso comprises both the resistance which is referred to as classicalresistance mediated through P glycoprotein, as well as the resistancereferred to as atypical resistance which is mediated by MRP or other,non-P-glycoprotein mediated resistances. A further marker whichcorrelates with the expression of YB-1 is topoisomerase II alpha.Insofar topoisomerase II alpha may be used in a screening method insteadof or in addition to determining YB-1 in the nucleus in order to decidewhether a patient may be treated in accordance with the invention usingthe adenoviruses with an expectation of success. A marker which, inprinciple, may be similarly used as the P glycoprotein, is MRP. Afurther marker, at least to the extent that colorectal carcinoma cellsor patients having colorectal carcinoma are concerned, is PCNA(proliferating cell nuclear antigen) (Hasan S. et al., Nature, 15,387-391, 2001), as, for example, described by Shibao K. et al. (Shibao Ket al., Int. Cancer, 83, 732-737, 1999). Finally, at least in the fieldof breast cancer and osteosarcoma cells, the expression of MDR (multipledrug resistance) is a marker in the afore-described meaning (Oda Y etal., Clin. Cancer Res., 4, 2273-2277, 1998). A further potential markerwhich may be used in accordance with the invention is p73 (Kamiya, M.,Nakazatp, Y., J Neurooncology 59, 143-149 (2002); Stiewe et al., J.Biol. Chem., 278, 14230-14236, 2003).

Finally, it shall also be referred to YB-1 as a prognostic marker inbreast cancer which may be used in the present invention. Only inpatients having increased expression of YB-1 in the primary tumor, arecurrence occurs after surgery and chemotherapy [Janz M. et al. Int. J.Cancer 2002, 97, 278-282].

It is a particular advantage of the present invention that also thosepatients may be subject to treatment using in accordance with theinvention the adenoviruses described herein, which otherwise cannot betreated anymore in the medicinal-clinical sense and where thus a furthertreatment of the tumor diseases using the methods of the prior art is nolonger possible with an expectation of success, in particular where theuse of cytostatics and irradiation is no longer reasonably possible andcannot be successfully carried out any longer in the sense ofinfluencing or reducing the tumor. Herein the term tumor refers ingeneral also to any tumor or cancer disease which either inherentlycontains YB-1 in the cellular nucleus, preferably independent of thecell cycle, or does so by applying exogenous measures, as disclosedherein, and/or which contains deregulated YB-1.

Additionally, the viruses described herein can, in principle, be usedfor the treatment of tumors.

The tumours which can in particular be treated by the viruses describedherein are preferably those tumours which are selected from the groupcomprising tumours of the nervous system, ocular tumours, tumours of theskin, tumours of the soft tissue, gastrointestinal tumours, tumours ofthe respiratory system, tumour of the skeleton, tumours of the endocrinesystem, tumours of the female genital system, tumours of a mammarygland, tumours of the male genital system, tumours of the urinaryoutflow system, tumours of the haematopoietic system including mixed andembryonic tumours. It is within the present invention that these tumoursare in particular resistant tumours as in particular defined herein.

The group of tumors of the nervous system preferably comprises:

-   -   1. Tumors of the skull as well as of the brain (intracranial),        preferably astrocytoma, oligodendroglioma, meningioma,        neuroblastoma, ganglioneuroma, ependymoma, schwannoglioma,        neurofibroma, haemangioblastoma, lipoma, craniopharyngioma,        teratoma and chordoma;    -   2. Tumors of the spinal cord and of the vertebral canal,        preferably glioblastoma, meningioma, neuroblastoma,        neurofibroma, osteosarcoma, chondrosarcoma, haemangiosarcoma,        fibrosarcoma and multiple myeloma; and    -   3. Tumors of the peripheral nerves, preferably schwannoglioma,        neurofibroma, neurofibrosarcoma and perineural fibroblastoma.

The group of the ocular tumors preferably comprises:

-   -   1. Tumors of the eyelids and of the lid glands, preferably        adenoma, adenocarcinoma, papilloma, histiocytoma, mast cell        tumor, basal-cell tumor, melanoma, squamous-cell carcinoma,        fibroma and fibrosarcoma;    -   2. Tumors of the conjunctiva and of the nictitating membrane,        preferably squamous-cell carcinoma, haemangioma,        haemangiosarcoma, adenoma, adenocarcinoma, fibrosarcoma,        melanoma and papilloma; and

3. Tumors of the orbita, the optic nerve and of the eyeball, preferablyretinoblastoma, osteosarcoma, mast cell tumor, meningioma, reticularcell tumor, glioma, schwannoglioma, chondroma, adenocarcinoma,squamous-cell carcinoma, plasma cell tumor, lymphoma, rhabdomyosarcomaand melanoma.

The group of skin tumors preferably comprises:

-   -   Tumors of the histiocytoma, lipoma, fibrosarcoma, fibroma, mast        cell tumor, malignant melanoma, papilloma, basal-cell tumor,        keratoacanthoma, haemangiopericytoma, tumors of the hair        follicles, tumors of the sweat glands, tumors of the sebaceous        glands, haemangioma, haemangiosarcoma, lipoma, liposarcoma,        malignant fibrous histiocytoma, plasmacytoma and lymphangioma.

The group of tumors of the soft-tissues preferably comprises:

-   -   Tumors of the alveolar soft-tissue sarcoma, epithelioid cell        sarcoma, chondrosarcoma of the soft-tissue, osteosarcoma of the        soft-tissues, Ewing's sarcoma of the soft-tissues, primitive        neuroectodermal tumors (PNET), fibrosarcoma, fibroma,        leiomyosarcoma, leiomyoma, liposarcoma, malignant fibrous        histiocytoma, malignant haemangiopericytoma, haemangioma,        haemangiosarcoma, malignant mesenchymoma, malignant peripheral        nerve sheath tumor (MPNST, malignant schwannoglioma, malignant        melanocytic schwannoglioma, rhabdomyosarcoma, synovial sarcoma,        lymphangioma and lymphangiosarcoma.

The group of gastrointestinal tumors preferably comprises:

-   -   1. Tumors of the oral cavity and of the tongue, preferably        squamous-cell carcinoma, fibrosarcoma, Merkel cell tumor,        inductive fibroameloblastoma, fibroma, fibrosarcoma, viral        papillomatosis, idiopathic papillomatosis, nasopharyngeal        polyps, leiomyosarcoma, myoblastoma and mast cell tumor;    -   2. Tumors of the salivary glands, preferably adenocarcinoma;    -   3. Tumors of the oesophagus, preferably squamous-cell carcinoma,        leiomyosarcoma, fibrosarcoma, osteosarcoma, Barrett carcinoma        and paraoesophageal tumors;    -   4. Tumors of the exocrine pancreas, preferably adenocarcinoma;        and    -   5. Tumors of the stomach, preferably adenocarcinoma, leiomyoma,        leiomyosarcoma and fibrosarcoma.

The group of the tumors of the respiratory system preferably comprises:

-   -   1. Tumors of the nose and nasal cavity, of the larynx and of the        trachea, preferably squamous-cell carcinoma, fibrosarcoma,        fibroma, lymphosarcoma, lymphoma, haemangioma, haemangiosarcoma,        melanoma, mast cell tumor, osteosarcoma, chondrosarcoma,        oncocytoma (rhabdomyoma), adenocarcinoma and myoblastoma; and    -   2. Tumors of the lung, preferably squamous-cell carcinoma,        fibrosarcoma, fibroma, lymphosarcoma, lymphoma, haemangioma,        haemangiosarcoma, melanoma, mast cell tumor, osteosarcoma,        chondrosarcoma, oncocytoma (rhabdomyoma), adenocarcinoma,        myoblastoma, small-cell carcinoma, non-small cell carcinoma,        bronchial adenocarcinoma, bronchoalveolar adenocarcinoma and        alveolar adenocarcinoma.

The group of the skeleton tumors preferably comprises:

-   -   osteosarcoma, chondrosarcoma, parosteal osteosarcoma,        haemangiosarcoma, synovial cell sarcoma, haemangiosarcoma,        fibrosarcoma, malignant mesenchymoma, giant-cell tumor, osteoma        and multilobular osteoma.

The group of the tumors of the endocrine system preferably comprises:

-   -   1. Tumors of the thyroid gland/parathyroid, preferably adenoma        and adenocarcinoma;    -   2. Tumors of the suprarenal gland, preferably adenoma,        adenocarcinoma and pheochromocytoma (medullosuprarenoma);    -   3. Tumors of the hypothalamus/hypophysis, preferably adenoma and        adenocarcinoma;    -   4. Tumors of the endocrine pancreas, preferably insulinoma (beta        cell tumor, APUDom) and Zollinger-Ellison syndrome (gastrin        secernent tumor of the delta cells of the pancreas); and    -   5. as well as multiple endocrine neoplasias (MEN) and        chemodectoma.

The group of the tumors of the female sexual system tumors preferablycomprises:

-   -   1. Tumors of the ovaries, preferably adenoma, adenocarcinoma,        cystadenoma, and undifferentiated carcinoma;    -   2. Tumors of the uterine, preferably leiomyoma, leiomyosarcoma,        adenoma, adenocarcinoma, fibroma, fibrosarcoma and lipoma;    -   3. Tumors of the cervix, preferably adenocarcinoma, adenoma,        leiomyosarcoma and leiomyoma;    -   4. Tumors of the vagina and vulva, preferably leiomyoma,        leiomyosarcoma, fibroleiomyoma, fibroma, fibrosarcoma, polyps        and squamous-cell carcinoma.

The group of tumors of the mammary glands preferably comprises:

-   -   fibroadenoma, adenoma, adenocarcinoma, mesenchymal tumora,        carcinoma, carcinosarcoma.

The group of the tumors of the male sexual system preferably comprises:

-   -   1. Tumors of the testicles, preferably seminoma,        interstitial-cell tumor and Sertoli cell tumor;    -   2. Tumors of the prostate, preferably adenocarcinoma,        undifferentiated carcinoma, squamous-cell carcinoma,        leiomyosarcoma and transitional cell carcinoma; and    -   3. Tumors of the penis and the external gentials, preferably        mast cell tumor and squamous-cell carcinoma.

The group of tumors of the urinary outflow system preferably comprises:

-   -   1. Tumors of the kidney, preferably adenocarcinoma, transitional        cell carcinoma (epithelial tumors), fibrosarcoma, chondrosarcoma        (mesenchymal tumors), Wilm's tumor, nephroblastoma and embryonal        nephroma (embryonal pluripotent blastoma);    -   2. Tumors of the ureter, preferably leiomyoma, leiomyosarcoma,        fibropapilloma, transitional cell carcinoma;    -   3. Tumors of the urinary bladder, preferably transitional cell        carcinoma, squamous-cell carcinoma, adenocarcinoma, botryoid        (embryonal rhabdomyosarcoma), fibroma, fibrosarcoma, leiomyoma,        leiomyosarcoma, papilloma and haemangiosarcoma; and    -   4. Tumors of the urethra, preferably transitional cell        carcinoma, squamous-cell carcinoma and leiomyosarcoma.

The group of tumors of the haematopoietic system preferably comprises:

-   -   1. Lymphoma, lymphatic leukemia, non-lymphactic leukemia,        myeloproliferative leukemia, Hodgkin's lymphoma, Non-Hodgkin's        lymphoma.

The group of the mixed and embryonal tumors preferably comprises:

-   -   Haemangiosarcoma, thymoma and mesothelioma.

In a particularly preferred embodiment these tumors are selected fromthe group comprising breast cancer, ovary carcinoma, prostate carcinoma,osteosarcoma, glioblastoma, melanoma, small-cell lung carcinoma andcolorectal carcinoma. Further tumors are those which are resistant asdescribed herein, preferably those which are multiple resistant,particularly also those tumors of the group described above. Especiallypreferred tumors are also those selected from the group comprisingbreast tumors, bone tumors, stomach tumors, intestinal tumors,gallbladder tumors, pancreatic tumors, liver tumors, kidney tumors,brain tumors, ovary tumors, tumors of the skin and of cutaneousappendages, head/neck tumors, uterus tumors, synovial tumors, larynxtumors, oesophageal tumors, tongue tumors and prostate tumors. It ispreferred that these tumors are those which are, regarding theirmanifestations, disclosed herein altogether.

The adenoviruses of the invention, preferably the group I adenovirusesand the adenoviruses to be used in accordance with the invention,preferably the group II adenoviruses.

The use of the adenoviruses disclosed herein, particularly group Iadenoviruses and/or group II adenoviruses as medicaments and inparticular in connection for systemic administration can be improved bya suitable targeting of the adenoviruses. The infection of tumor cellsby adenoviruses depends, among others, to a certain extent on thepresence of the coxackievirus-adenovirus receptor CAR and distinctintegrins. As soon as they are strongly expressed in cells, preferablytumor cells, an infection is possible already at very low titers(pfu/cell). Various strategies have been tried to date in order to reacha so-called re-targeting of recombinant adenoviruses, for example byinserting heterologous sequences in the fiber knob region, usingbi-specific antibodies, coating of the adenoviruses with polymers,introducing ligands in the Ad fiber, substituting the serotype 5 knoband serotype 5 fiber shaft and knop by the serotype 3 knob and Ad 35fiber shaft and knob, and modification of the penton base, respectively(Nicklin S. A. et al., Molecular Therapy 2001, 4, 534-542; Magnusson, M.K. et al., J. of Virology 2001, 75, 7280-7289; Barnett B. G. et al.,Biochimica et Biophysica Acta 2002, 1575, 1-14). The realisation inconnection with the various aspects of the present invention of suchfurther embodiments and features, respectively, in the adenoviruses inaccordance with the invention and the adenoviruses used in accordancewith the invention, particularly in group I adenoviruses and group IIadenoviruses, is within the present invention.

The invention is related in a further aspect to a method for thescreening of patients which may be treated using a modified adenovirus,i.e. an adenovirus as used in accordance with the invention, such as,for example, AdΔ24, dl922-947, E1Ad/01/07, CB016 or the virusesdescribed in European patent EP 0 931 830, and/or a group I adenovirusand/or group II adenovirus, whereby the method comprises the followingsteps:

-   -   Analysing a sample of the tumor tissue and    -   Determining whether YB-1 is localised in the nucleus independent        of the cell cycle, or whether the cells contain deregulated        YB-1.

Instead of or in addition to YB-1 also the presence of theafore-described markers can be assessed.

In case that the tumor tissue or a part thereof comprises YB-1 in thenculeus, preferably independent of the cell cycle, or comprisesderegulated YB-1, the adenoviruses as disclosed herein, particularlygroup I adenoviruses and/or group II adenoviruses may be used inaccordance with the present invention.

In an embodiment of the method according to the invention it iscontemplated that the analysis of the tumor tissue occurs by means of anagent which is selected from the group comprising antibodies againstYB-1, aptamers against YB-1, spiegelmers against YB-1 as well asanticalines against YB-1. In principle, the same kind of agents can alsobe made and used, respectively, for the respective markers. Themanufacture of antibodies, in particular monoclonal antibodies, is knownto the ones skilled in the art. A further agent for specific detectionof YB-1 or the markers are peptides which bind with a high affinity totheir target structures, in the present case YB-1 or said markers. Inthe prior art methods are known such as, for example, phage-display, inorder to generate such peptides. For such purpose, it is started from apeptide library whereby the individual peptides have a length of about 8to 20 amino acids and the size of the library is about 10² to 10¹⁸,preferably 10⁸ to 10¹⁵ different peptides. A particular form of targetmolecule binding polypeptides are the so-called anticalines which are,for example, described in German patent application DE 197 42 706.

A further agent for specifically binding to YB-1 or the correspondingmarkers disclosed herein and thus for the detection of a cell cycleindependent localisation of YB-1 in the nucleus, are the so-calledaptamers, i.e. D-nucleic acids, which, based on RNA or DNA, are presentas either a single strand or a double strand and specifically bind to atarget molecule. The generation of aptamers is, for example, describedin European patent EP 0 533 838. A special embodiment of aptamers arethe so-called aptazymes which, for example, are described by Piganeau,N. et al. (2000), Angew. Chem. Int. Ed., 39, no. 29, pages 4369-4373.They are a particular embodiment of aptamers insofar as they compriseapart from the aptamer moiety a ribozyme moiety and, upon binding orrelease of the target molecule binding to the aptamer moiety, theribozyme moiety becomes catalyctically active and cleaves a nucleic acidsubstrate which goes along with generation of a signal.

A further form of the aptamers are the so-called spiegelmers, i.e.target molecule binding nucleic acids which consist of L-nucleic acids.The method for the generation of such spiegelmers is, for example,described in WO 98/08856.

The sample of the tumor tissue can be obtained by punctuation orsurgery. The assessment whether YB-1 is located in the nucleusindependent of the cell cycle is frequently done by the use ofmicroscopic techniques and/or immunohistoanalysis, typically using theantibody or any of the further agents described above. Further methodsfor the detection of YB-1 in the nucleus and that its localisation thereis independent of the cell cycle, are known to the one skilled in theart. For example, localisation of YB-1 can easily be detected whenscanning tissue slices stained against YB-1. The frequency of YB-1 beingin the nucleus is already an indication that the localisation in thenucleus is independent of the cell cycle. A further possibility for cellcycle independent detection of YB-1 in the nucleus is the stainingagainst YB-1 and assessment whether YB-1 is localised in the nucleus anddetermining the phase of the cells. This and the detection of YB-1,respectively, however, can also be performed using the afore-mentionedagents directed against YB-1. The detection of the agents is done byprocedures known to the one skilled in the art. Because said agents arespecifically directed against YB-1 and insofar do not bind to otherstructures within the sample to be analysed, particularly otherstructures of the cells, both the localisation of said agents by meansof a suitable labelling of the agents and due to their specific bindingto YB-1, also the localisation of YB-1 can be detected and assessedaccordingly. Methods for the labelling of the agents are known to theones skilled in the art. The same techniques may also be used in orderto determine whether and if so how many of the cells of the samplecontain deregulated YB-1. As deregulated YB-1 also shows anoverexpression compared to non-deregulated YB-1, the relative expressionof YB-1 compared to a reference sample may be used in order to determinewhether YB-1 is deregulated in the analysed cell.

It is within the present invention that the viruses described herein,whether they are the viruses of the present invention or whether theyare the viruses to be used in accordance with the present invention, arealso used in connection with diseases, preferably tumor diseases andmore preferably tumor diseases where at least a part of the tumor cellsexhibits a multiple resistance, in particular a multi-drug resistance,in which YB1 is deregulated. This applies also to each and any otheraspect as described herein in connection with the cells and tumors tothe extent that it refers to cells and diseases where YB1 is present inthe nucleus, preferably independent of the cell cycle.

The present invention shall now be further illustrated using the figuresand examples, whereby novel features, embodiments and advantages of theinvention may be taken therefrom. In connection therewith

FIG. 1 shows the structural design of the adenoviral vectors referred totherein as AdE1/E3-minus adenoviral vectors which are E1/E3-deletedadenoviruses, of wildtype adenovirus and of adenovirus dl520;

FIG. 2 shows the binding domains of the E1A proteins with respect to thebinding of p300, p107 and p105;

FIG. 3 shows U2OS cells which do not have YB-1 in the nucleus, afterinfection with the E1/E3-deleted adenovirus Ad5 referred to therein asE1/E3-minus Ad5, and dl520;

FIG. 4 shows 257RDB cells which contain YB-1 in the nucleus, afterinfection with the E1/E3-deleted adenovirus Ad5 referred to therein asE1/E3-minus Ad5, and adenovirus dl520;

FIG. 5 shows 257RDB cells and U2OS cells after infection with adenovirusdl 119/1131;

FIG. 6 shows the result of an EMSA analysis which confirms that YB-1 ispresent in the cellular nucleus in multi-resistant cells and in celllines 257RDB, 181 RDB, MCF-7Ad, whereas YB-1 is not present in thenucleus of US2OS and HeLa cells;

FIG. 7 shows the structural design of the E1A protein of wildtypeadenovirus, of adenovirus dl520 and of adenovirus dl119/1131;

FIG. 8 shows a bar graph indicating replication efficiency of adenovirusin the presence of additionally expressed viral proteins in absolutefigures;

FIG. 9 shows a bar graph indicating the increase in replicationefficiency of adenoviruses in the presence of additionally expressedviral proteins;

FIG. 10 shows wells with U2OS cells grown therein after crystal violetstaining and infection with dl520 with 10 and 30 pfu/cell and control(K), respectively, without administration of daunorubicin and withadministration of 40 ng daunorubicin per ml;

FIG. 11 shows wells having HeLa cells grown therein after crystal violetstaining and infection with dl520 with 10 and 30 pfu/cell and control(K), respectively, without administration of daunorubicin and withadministration of 40 ng daunorubicin per ml;

FIG. 12 shows a diagram of the tumor volume as a function of time oftumors of different origin (RDB257 and HeLa) after treatment with PBSand dl520, respectively;

FIG. 13 shows pictures of sacrificed mice which developed a tumor basedon RDB257 cells, after treatment with PBS and 5×10⁸ pfu dl520,respectively;

FIG. 14 shows the result of a Southern blot analysis of a cell extract(of subcutaneously grown tumors) of RDB257 cells and HeLa cells afterinfection with dl520;

FIG. 15 shows a bar graph indicating replication efficiency and particleformation of dl520 and wildtype adenovirus in YB-1 nucleus-positivetumor cells (257RDB and 181RDB) and YB-1 nucleus-negative tumor cells(HeLa, U2OS);

FIG. 16 shows the structural design of wildtype adenovirus and theadenoviral vector AdXVir03;

FIG. 17 shows the structural design of the adenoviral vectorAdXVir03/01; and

FIG. 18A/B shows wells having grown 18RDB cells (FIG. 18A) and 272RDBcells (FIG. 18B) after crystal violet staining and infection with Ad312(20 pfu/cell), Xvir03 (5 pfu/cell) and control (non-infected), wherebycrystal violet staining was performed five days after infection;

FIG. 19 shows the result of a Northern blot analysis of the expressionof the E2 gene in A549 cells and U2OS cells after infection withwildtype adenovirus Ad5 and adenovirus Ad312;

FIG. 20 shows the result of a Northern blot analysis of the expressionof the E2 gene in U2OS cells after infection with wildtype adenovirusand adenovirus delta24 after 12 and 24 hours;

FIG. 21 shows the structural design of the adenoviral vector XvirPSJL1;

FIG. 22 shows the structural design of the adenoviral vector XvirPSJL2;

FIG. 23 shows wells with HeLa cells grown therein after crystal violetstaining and infection with adenovirus dl520 using different pfu/cells;

FIG. 24 shows a bar graph indicating the activity of luciferase in U2OScells, HeLa cells and 257RDB cells upon usage of different promoterfragments of the adenoviral E2-late promoter;

FIG. 25 shows a bar graph indicating the number of viral particles afterinfection of U2OS cells with a YB-1 expressing adenovirus and virusAd312 after two and five days, whereby a distinction is made betweenintracellularly remaining viral particles (represented in black) andreleased extracellular viral particles (horizontally striped);

FIG. 26 shows the result of a Southern Blot analysis of the replicationbehaviour of Adenovirus dl 520 in U373 cells with and without treatmentof the cells with irinotecan;

FIG. 27 shows the result of a Southern Blot analysis of the replicationbehaviour of adenovirus dl 520 in U 373 cells with and without treatmentof the cells with trichostatin A;

FIG. 28 shows the result of a FACS analysis of U 373 cells treateed withtrichostatin for the expression of Coxsackievirus-Adenovirus receptor(CAR), expressed as percentage of CAR-psotive cells;

FIG. 29 shows four different panels of cell layer for the illustrationof the effecto of replicating adenovirus dl520 and irinotecan andtrichostatin in different combinations;

FIG. 30 shows a schematic representation of the ORF of E1B55K with the3′UTR fragment and the restriction site BfrI at position 3532;

FIG. 31 shows the sequence of the E1B55k-3′UTR region correspondingsequence position 3507 to 4107 of wildtype Ad5; and

FIG. 32 shows a schematic representation of a universal shuttle plasmidfor the generation of E3/E4 modified recombinant adenoviruses having theRGD motif.

EXAMPLE 1 Types of E1A Modifications as May be Comprised by theAdenoviruses Which are Used in Accordance with the Invention

FIG. 1 shows the structural design of adenoviral vectors AdE1/E3-minus,i.e. E1/E3-deleted adenoviruses, wildtype adenovirus and adenovirusdl520.

Adenovirus AdE1/E3-minus does not have a region coding for a functionalE1A or a functional E1B or E3 and is used in the present experiments asa control for toxicity.

Wildtype E1A gene codes for a total of 5 proteins which are generatedthrough alternative splicing of the E1A RNA. Among others, two differentproteins are generated, namely a 289 amino acid protein and a 243 aminoacid protein. dl520 does not code for the 289 amino acid protein as ithas a deletion in the CR3 stretch of the E1A gene which results in thelack of the 13S gene product. The adenovirus dl520 which may be used inaccordance with the invention is referred to as 12S-E1A virus by thoseskilled in the art. Adenovirus dl347 (Wong und Ziff, J. Virol., 68,4910-4920, 1994) known in the prior art is also a 12S-E1A virus whichcan be used in accordance with the present invention.

Within the 289 amino acid protein which is encoded by the 13S-E1A mRNA,there are 3 regions which are conserved among various adenoviralsubtypes. These are referred to as CR1, CR2 and CR3. While C R1 and CR2are present in both E1A proteins (E1A 12S and E1A 13S), i.e. in both the289 amino acid and the 243 amino acid protein, the CR3 region is onlypresent in the bigger one of the two aforementioned proteins.

The CR3 region is required for the activation of viral genes, inparticular of E1B, E2, E3 and E4. Viruses which only comprise thesmaller, i.e. 243 amino acid protein are only very weaklytransactivating the viral genes and do not promote adenoviralreplication in those cells which do not have YB-1 in the nucleus. AsYB-1 is present in the nucleus only in tumor cells and can be detectedonly there, this vector is suitable to induce tumor-specificreplication.

Due to the deletion of CR3 in dl520 this adenovirus cannot translocatecellular YB-1 into the cell's nucleus which is also referred to hereinas translocation, and is thus not in a position to replicate in cellswhich are YB-1 nucleus-negative and is thus a virus which can be used inaccordance with the present invention, whereby this virus comprises thetransactivation required in accordance with the present invention.

EXAMPLE 2 Mode of Action of Adenoviruses in Depending on the Rb Statusof Cells

FIG. 2 shows the binding domains of the E1A protein with regard to thebinding of p300, p107 and p105. P300, as well as p107, is a cellularbinding protein. The binding of the retinoblastoma protein (pRb), atumor suppressor protein, is mediated through CR1 and CR2. Studies haveshown that pRb and p107/p300 are in combination with the cellulartranscription factor E2F effective in regulating transcription. Thewildtype E1A protein interferes with the binding of E2F to Rb. The thusreleased E2F binds to the E2 early promoter and induces adenoviralreplication thereby.

It is known from the prior art that certain deletions in the E1Aoncoprotein may result in recombinant adenoviral vectors such as thosementioned in the following, which are capable of replicatingpredominantly in Rb-negative cells and can be used in accordance withthe present invention. For example, the adenoviral vector dl922-947comprises a deletion in the CR2 region (amino acid positions 122-129)and the vector CB016 has deletions in the CR1 region (amino acidpositions 27-80) and CR2 region (amino acid positions 122-129). Thevector E1Adl01/07 comprises a deletion in the CR2 region (amino acidpositions 111-123). Additionally, because of an additional deletion atthe N-terminus (amino acid positions 4-25), additionally, there is nobinding to protein p300. The adenoviral vector AdΔ24 comprises adeletion in the CR2 region (amino acid positions 120-127). Theadenoviral vector described in patent EP 0 931 830 comprises deletionsin the CR1 region and CR2 region.

The binding mechanism of E2F/RB and the release of E2F mediated throughE1A is fundamentally different from the mechanism underlying the presentinvention. Unlike assumed in the prior art it is not the release of E2Ffrom the Rb protein which is essential, not to say critical for viralreplication, but it is the nuclear localisation of the humantranscription factor YB-1. This transcription factor is, in normalcells, only present in the cytoplasm over most of the cell cycle. Afterinfection with an adenovirus it is induced into the nucleus undercertain circumstances or is already present in the nucleus in distinctcellular systems, such as distinct tumor diseases including, forexample, but not limited thereto, breast cancer, ovary carcinoma,prostate carcinoma, osteosarcoma, glioblastoma, melanoma, small celllung carcinoma and colorectal carcinoma.

EXAMPLE 3 Infection of U2OS Cells

100,000 U2OS cells were plated per well. On the next day the cells wereinfected with the various adenoviruses as depicted in FIG. 3. Theinfection was performed in 500 μl serum free DMEM medium at 37° C. for 1h. Subsequently, the infection medium was removed and replaced by 2 mlcomplete medium (10% FCS/DMEM). The analysis was performed after 3 daysusing crystal violet staining.

As may be taken from FIG. 3, the U2OS cells which do not have YB-1 inthe nucleus, show no lysis as illustrated by crystal violet stainingafter infection with two different adenoviruses, namely theE1/E3-deleted adenovirus referred to as E1/E3-minus, and adenovirusdl520, which can be used in accordance with the present invention. Inconnection therewith, first, the medium is removed. Subsequently, thecells are overlaid with crystal violet (50% ETOH, 3% formaldehyde, 5%acetic acid, 1% crystal violet) and incubated at room temperature for5-10 min. Subsequently, the plates having 6 wells are thoroughly rinsedwith water and dried at room temperature.

This confirms the finding underlying the present invention that thepresence of YB-1 is required in order to induce the viruses used inaccordance with the present invention, to lyse the infected cells.

EXAMPLE 4 Infection of 257RDB Cells

100,000 257RDB cells were plated per well. On the next day the cellswere infected with the various adenoviruses as depicted in FIG. 4. Theinfection was performed in 500 μl serum free DMEM medium for 1 h at 37°C. Subsequently, the infection medium was removed and replaced by 2 mlcomplete medium (10% FCS/DMEM). The analysis was performed after threedays using crystal violet staining.

The result of this experiment is depicted in FIG. 4. The adenovirusreferred to as E1/E3-minus Ad5 which is E1/E3-deleted, did not show anylysis at low MOIs (pfu/cell) upon infection of 257RDB cells which haveYB-1 in the nucleus. In contrast thereto, dl520 which, as shown inexample 3, does not replicate in YB-1 nucleus-negative cells and at thesame time codes with E1A for a transactivating oncogene protein inaccordance with the present invention, results in a factually completelysis at an MOI (multiplicity of infection) of 40 pfu per cell and astill predominant lysis at an MOI of 10 pfu per cell. It can beconcluded therefrom that dl520 and similar viruses such as describedherein by dl1119/1131 or AdXvir 03, require an MOI which is reduced byabout 1 magnitude (factor of ten) compared to E1-deleted or anE1/E3-deleted adenovirus which justifies their clinical use.

As depicted in FIG. 7, the protein E1A of dl520 is characterised in thatthe CR3 region thereof is deleted which results in the transactivationrequired for the use in accordance with the present invention andreplication in YB-1 nucleus-positive cells.

EXAMPLE 5 Infection of 257RDB and U2OS Cells with dl1119/1131

As depicted in FIG. 5, there is no lysis at an MOI of 20 pfu per cellupon infection of YB-1 nucleus-negative U2OS cells with adenovirusdl1119/1131 which exhibits a deletion of amino acids 4-138 of the E1Aprotein and the nucleic acid coding therefor, and further comprises astop codon after amino acid 218, whereby the expressed truncated E1Aprotein comprises the CR3 region of the complete E1A protein. As anegative control a non-infected cell layer was used.

In contrast thereto, there was factually a complete lysis of the celllayer at an MOI of 20 pfu per cell under the influence of adenovirusdl1119/1131 in a cellular system such as 257RDB which contains YB-1 inthe nucleus, i.e. is YB-1 nucleus-positive. Insofar this example isanother proof that a modified E1A oncogene protein which, as depicted inFIG. 7, comprises, for example, only the CR3 region and which is lackingthe CR1 region and CR2 region, provides for the required transactivationin YB-1 nucleus-positive cells which is required for the replication ofadenoviruses in accordance with the present invention, which results inviral replication. The adenovirus dl1119/1131 is thus a furtheradenovirus which can be used in accordance with the present invention.It is within the present invention that also viruses can be used whichare designed similar to dl1119/1131 with regard to the CR3 region, but,in contrast thereto, have the CR1 region and/or CR2 region.

EXAMPLE 6 Detection of Nuclear YB-1 in Multidrug Resistant Cells

The example is based on the consideration that nuclear YB-1 should bindas a transcription factor to the Y-box (CAAT sequence) within the mdr1promoter (engl. multiple drug resistance promoter). In order to detectthis, a so-called EMSA analysis (electrophoretic mobility shift assay)was performed. In connection therewith, nuclear protein is isolated andsubsequently 1-10 μg protein is incubated together with a short DNAfragment (oligo) at 37° C. In order to determine nuclear YB-1, thefollowing oligonucleotide was used: mdr1 promoter in contrast to U203(Position −86 to −67): TGAGGCTGATTGGCTGGGCA (the X-box is underlined).

This DNA fragment is radioactively labelled at the 5′ end with ³²P priorto that. Subsequently, separation is performed in a native polyacrylamide gel. In case the protein YB-1 is binding to a sequence in theoligonucleotide, this can be detected as any non-bound oligonucleotideis migrating faster in the gel than bound oligonucleotide (Holm, P. S.et al., JBC 277, 10427-10434, 2002; Bargou, R. C. et al., NatureMedicine 3, 447-450, 1997).

As depicted in FIG. 6, it could be shown with the EMSA analysis thatYB-1 is present in the nucleus of multidrug resistant cells 257RDB,181RDB and MCF-7Ad cells in contrast to cell lines U2OS and HeLa cells.

The results shown in example 4 and 5 confirm that the adenoviruses dl520and dl1119/1131 replicate in YB-1 nucleus-positive cells such as, e.g.,257RDB in contrast to U205, and induce lysis thereof. This confirms thefinding about the use of the adenoviruses in accordance with the presentinvention. Additionally, the results confirm that already a, compared towildtype adenovirus, weak transactivation of viral genes in YB-1nucleus-positive cells through modified or deleted E1A gene productsresults in successful replication and lysis of such cells in thepresence of YB-1 in the nucleus, including, for example, multidrugresistant cells and that the adenoviruses as described herein, can thusbe used in the lysis of such tumors.

EXAMPLE 7 Increase of Replication Efficiency of E1-Minus Adenoviruses

This example shows that the early viral genes E1B-55K and E4orf6 can besubstituted through transfection with the plasmid pE4orf6 and infectionwith the E1/E3-deleted adenovirus Ad-55K. Ad-55K is an E1/E3 deletedvirus, whereby E1B-55K is cloned into E1 and is under the control ofCMV. This substitution is necessary with regard to the fact that AdYB-1,i.e. an adenovirus which expresses YB-1, does not express these earlygenes and that the present inventor has recognised that a substitutionof these early genes in a replication system which contains YB-1 in thenucleus, is capable of increasing replication efficiency and particleformation efficiency, respectively, to an extent comparable to the oneof wildtype adenoviruses of type Ad5.

The following was done:

Transfection of each 10⁵ U2OS cells with the plasmid pE4orf6 usinglipofectamine. The plasmid pE4orf6 carries the DNA sequence coding forthe early viral gene E4orf6 under the control of CMV.

24 h after transfection with the plasmid pE4orf6 the cells were infectedwith the YB-1 expressing E1/E3-deleted adenovirus AdYB-1 (50 pfu/cell)and the E1/E3-deleted E1B-55K adenovirus Ad-55K (50 pfu/cell). Ad-55K isan E1/E3-deleted virus which carries as transgene the viral gene E1B-55Kunder CMV control.

Subsequently, the cells were removed from the medium (2 ml) 5 days afterinfection (=post infectionem). The release of the viral particles fromthe isolated cells was done by alternating freezing and thawing forthree times (thaw/freeze). Subsequently, a plaque assay was performed on293 cells for determining the generated infectious particles (plaqueforming units per ml (pfu/ml)). The result is depicted in FIGS. 8 and 9.FIG. 8 shows the result of the plaque assay, represented in absolutefigures. The most significant difference compared to infection withAdYB-1 alone is shown by transfection with the plasmid pE4orf6 andco-infection with the two viruses AdYB-1 and Ad-55K. FIG. 9 shows theresult of FIG. 8, whereby the increase of the replication efficiency isrepresented as multifold of the replication determined for AdYB-1. Thecells infected with plasmid pE4orf6 and subsequently with AdYB-1 andE1B-55K (Ad-55K) produced up to 25 times more pfu/ml.

Based on these results it can be concluded that the substitution ofE1B-55K and E4orf6 increases the number of viruses formed (pfu/ml) afterinfection with the E1/E3-deleted adenovirus AdYB-1 by a factor of up to25. The additive effects of E1B-55K and E4orf6 on the production ofplaque forming units (pfu) is significantly higher compared to theeffects of each of the two gene products.

Control experiments with one plasmid which expresses EGFP, clearlyshowed that in the experimental approach chosen only 10% of the cellswere successfully transfected with plasmid pE4orf6. The number of theparticles formed in the cells which express both E1B-55K and E4orf6 iscomparable to the one of human adenovirus type 5 (wildtype). Thisconfirms the finding underlying the present invention that theexpression of E4orf6 and E1B-55K is, in combination with the nuclearlocalisation of YB-1, able to provide for adenoviral replication andparticle formation, in particular of E1A-deleted adenoviruses, which iscomparable to the one of wildtype Ad5.

EXAMPLE 8 Increased Replication of Adenoviruses which are notReplicating in YB-1 Nucleus-Negative Cells, in YB-1 Nucleus-PositiveCells Upon Administration of Cytostatics

It is known in the prior art that the addition of different cytostaticsinduces nuclear localisation of the human transcription factor YB-1. Ashas been found by the present inventor, YB-1 localised in the nucleuscontrols adenoviral replication by means of activation of the adenoviralE2-late promoter. The combination of both effects can be used in orderto provide for specific tumor lysis.

In the practising of the oncolytic assays the following procedure wasfollowed: 200,000 cells (HeLa and U2OS, respectively) were plated intoeach well of a 6 well plate. On the next day 40 ng/ml (finalconcentration) of daunorubicine were added. After 3 hours of incubationthe cells were infected with 10 and 30 pfu dl520/cell, respectively.Subsequently, the cells were incubated in cytostatic free medium. After3-5 days the cells were stained using crystal violet.

As may be taken from FIGS. 10 and 11, the addition of daunorubicineinduces the replication of dl520 through nuclear localisation of YB-1.Thus, dl520 creates a bigger tumorlytic effect in combination with thecytostatic daunorubicine compared to daunorubicine alone.

EXAMPLE 9 In Vivo Tumor Lysis by dl520

The HeLa (YB-1 nucleus-negative) and 257RDB (YB-1 nucleus-positive)cells used in this in vivo study, were expanded under sterile cellculture conditions. Prior to the injection of the cells into mice(strain CD1NuNu) in order to generate a subcutaneous tumor, the cellsare harvested by trypsinisation, taken up in DMEM medium (10% FCS),counted and washed with PBS one time. Subsequently, the cells arecentrifuged, the PBS aspired and the cells are portioned in fresh PBSwith the desired cell number. The cell number which was subcutaneouslyinjected in this study, was each 5×10⁶ cells of both cell lines. Theinjection was performed subcutaneously into one flank of the animals,whereby HeLa cells were injected into the right side and 257RDB cellswere injected into the left side for better distinction. The growth ofthe tumors was controlled twice a week and thereby the length and thewidth of the tumors was measured using vernier calipers. Based thereon,the tumor volume was calculated based on the following mathematicalformula:3/4π*a/2*(b/2)² a=length, b=width

Once the tumor has reached a volume of 200 to 520 mm³, the virus and PBSas negative control, respectively, were intratumorally applied. Thevolumes to be injected were identical and were 50 μl each time. This wasrepeated on 3 consecutive days. The overall dosage of applied viruseswas 5×10⁸ pfu. Subsequently, the tumor growth was continued to bedocumented twice a week and the volume was calculated. At the end of thestudy the mice were sacrificed and the tumors removed for furtheranalysis.

The results are depicted in FIGS. 12 and 13.

FIG. 12 shows a diagram representing the tumor volume as a function oftime and the various treatment schemes. In case the tumor was formed byRDB257, there was a significant growth of the tumor to about 438 mm³ to1466 mm³ upon injection of PBS. Under the influence of the vector dl520which was used in accordance with the invention, tumor growth could bereduced significantly. Starting from a mean tumor size of 344 mm³, thetumor size increased only by 21% to a total of 543 mm³.

In the present example the tumor consisting of HeLa cells was used as acontrol which upon administration of PBS behaved similarly to the RDB257based tumor upon administration of PBS. Tumors based on HeLa cells andtreated with dl520, however, still showed a significant increase intumor growth starting from 311 mm³ and increasing to 1954 mm³.

FIG. 13 shows a picture of the sacrificed nude mice which had a tumorgrown using RDB257.

It can be clearly seen that after the application of adenovirus dl520 inaccordance with the present invention a significant reduction of thetumor occurred. In the present case there was even a reduction in thetumor volume (day 1 after administration of virus dl520: 515 mm³; day 30after administration of virus dl520: 350 mm³).

EXAMPLE 10 Southern Blot of Tumor DNA

DNA was extracted from a tumor sample which has been taken from themiddle of the tumor developed in example 9. For isolation the DneasyTissue Kit of Qiagen is used. The DNA isolation is done in accordancewith manufacturer's instructions. In accordance therewith, the DNA wasreleased from the cells through alkaline lysis. Subsequently, theisolated DNA is purified over a column. Subsequently, the concentrationof the isolated DNA is determined by photometry at 260 nm. The analysiswas performed using 2 μg of the DNA samples which were digested with 10units of restriction enzyme Kpn I. Subsequently, an electrophoreticseparation of the samples was performed in a 0.8% agarose gel.Subsequently, the DNA was blotted onto a nylon membrane (performedaccording to the system of Schleicher & Schuell). The DNA blotted ontothe membrane is hybridised against a specific 1501 bp DNA probe. The1501 bp DNA probe specifically binds to the 3369 bp Kpn I fragmentwithin the E2A coding Ad5 sequence. The probe was prepared prior to thatby PCR (primer: 5′-GTC GGA GAT CAG ATC CGC GT (SEQ. ID. No. 2), 5′-GATCCT CGT CGT CTT CGC TT (SEQ. ID. No.3)) and radioactively labelled using³²P. Subsequently, the membrane is washed and exposed to a film.

The result of the Southern Blot of tumor DNA is depicted in FIG. 14. Theanalysis confirms that only dl520 replicates in vitro in resistant cellsRDB257, as depicted in lanes 3, 4 and 5. Lane 1 shows as positivecontrol Ad-5d, lane 6, 7 and 8 show DNA from HeLa cells which wereinfected with dl520. As HeLa cells are not YB-1 nucleus positive thevirus dl520 did not replicate so that, in accordance therewith, the E2Asequence could not be detected.

A further result with dl520 is depicted in FIG. 15. Based on a plaqueassay the particle formation (pfu/ml) was investigated after infectionwith dl520 and wildtype adenovirus. Various YB-1 nucleus-positive(257RDB and 181RDB) tumor cells and YB-1 nucleus-negative tumor cellswere infected with dl520 and wildtype adenovirus.

The following procedure was practiced:

100,000-200,000 cells each were plated in so-called plates having 6wells (engl. 6 well plates) in L 15 medium (resistant cells) and DMEM(non-resistant cells) having 10% FCS. After 24 h infection with dl520and wildtype adenoviruses (10 pfu/cell) was performed. 3 days afterinfection (post infectionem) the viral particles were released from thecell suspension (3 ml) by alternating freezing and thawing for threetimes. Subsequently, a plaque assay was performed on 293 cells fordetermining the formed infectious particles (plaque forming units per ml(pfu/ml)). The result is depicted in FIG. 15. The result of the plaqueassay shows that dl520 is replicating in YB-1 nucleus-positive cells(257RDB and 181RDB) similar to wildtype adenovirus. Insofar areplication efficiency can be observed similar to the one of wildtypeadenoviruses when using, in accordance with the present invention, theadenoviruses described herein.

EXAMPLE 11 Structural Design of the Adenoviral Vector Xvir03

FIG. 16 shows the structural design of the adenoviral vector Xvir03. Theadenovirus Xvir03 is a so-called E1/E3-deleted adenovirus. This meansthat no E1A, E1B (E1B55k and E1B19K proteins) and E3 proteins aremanufactured which are functional in adenoviral replication. Thedeletion of the E1 region extends from 342-3528; the deletion of the E3region of the base position 27865-30995. As used herein, the term“E1-deleted virus” means a virus in which E1 is no longer functionallyactive. This can be achieved by inactivation with an otherwise mostlyintact nucleic acid and amino acid sequence, respectively, however, canalso mean a deletion of the E1 region coding proteins having varioussizes. Because of the lack of the E1A and E1B protein and the nucleicacids coding therefor, the E4 region, such as E4orf6, is only weaklyexpressed (about 1-5% compared to wildtype adenoviruses) or expressednot at all. The viral genes E1B55k and E4orf6 are expressed in the E1region by means of the heterologuous CMV promoter (Clontech: PlasmidpShuttle) introduced into Xvir03. Instead of the CMV promoter each andany of the promoters as disclosed herein in connection with theexpression of E1A can be used. The open reading frames of both genes arelinked with each other by means of a so-called IRES sequence (engl.internal ribosomal entry site) (Pelletier, J. and Sonenberg, N. Nature,1988, 334, 320-325). This element (Novagen: pCITE) provides for theexpression of 2 proteins from one mRNA.

The Vector was Manufactured as Follows: System Adeno-X of the CompanyClontech

The plasmid E1B55k-pShuttle was created by cloning the open readingframe of E1B55k from pCGNE1B from M. Dobelstein (University of Marburg)with XbaI and BfrI into the pShuttle vector from Clontech.Alternatively, the BamH1 fragment from the pCGNE1B vector can, afterhaving been made blunt ended, cloned into the correspondingly preparedpShuttle vector of Clontech. Subsequently, E1B55k in pShuttle waslinearised with ApaI, the ends blunt ended and cut with NheI.

In a second vector, pcDNA3.1 (+) (Invitrogen), subsequent to each other,the IRES element as a PCR product was cloned with pCITE-4a(+) of thecompany Novagen as template by means of TA cloning into the EcoRVcleaving site, and the E4orf6 from the plasmid pCMV-E4orf6 (M.Dobelstein, University of Marburg) was cloned by means ofBamHI=IRES-E4orf6-pcDNA3.1(+). IRES-E4orf6 in pcDNA3.1(+) was linearisedwith NotI, the ends blunt ended and subsequently the fragmentIRES-E4orf6 was cut out with NheI. The fragment IRES-E4orf6 was linkedwith the open vector E1B55k-pShuttle (blunt, NheI). The cassette wassubsequently cloned from the E1B55k-IRES-E4orf6-pShuttle together withthe CMV promoter and the bovine growth hormone (BGH)-PolyA into the ΔE1,ΔE3 Adeno-X-Plasmid (Clontech) with I-Ceu I and PI-SceI, and referred toas AdcmvE1B/IRES/E4orf6. Subsequently, the adenovirus was prepared inaccordance with manufacturer's instructions (Clontech). The adenoplasmid which was linearised with PacI having the expression elementCMV-E1B55k-IRES-E4orf6-BGH polyA was transfected into HEK293 cells and11 days post transfectionem the ablating cells were removed togetherwith the medium in order to release the generated adenoviruses throughrepeated freeze-thaw cycles.

It is within the present invention and feasible for the one skilled inthe art with regard to the technical teaching provided herein, thatother systems such as the system AdEasy of QBIOGENE and Microbix may beused for the manufacture of the adenoviruses according to the presentinvention, preferably the recombinant adenovirus, in particular thosewhich contain, individually and/or together, the cassettesE4orf6-IRES-E1B55k and YB-1-IRES-E1A12S. Additionally, individualtransgenes may be exchanged between the cassettes. It is within thepresent invention that also such adenoviruses can be manufactured andused in accordance with the present invention, where the cassette hasthe following design: E1B55k-IRES-E4orf6 and E1A12S-IRES-YB1.

In connection with the present invention a so called E1/E3 deletedrecombinant adenovirus was used which contains the cassetteE4orf6-IRES-E1B55k. It is, however, within an embodiment that the viruscomprises only an E1-deletion, which means that the E3-region remainsintact. Optionally, the E4-region may be partially and/or completelydeleted.

In the manufacture of the vector using different systems it wasproceeded as follows.

Manufacture of the adenovirus Ad-Xvir 3′UTR having an intact E3-regionwith the vector system according to Graham (company Microbix).

Cloning of the Vector CMV-E4ORF6-IRES-E1B55k 3′UTR-polyA in pDeltaE1sp1A

For the plasmid E1B55k 3′UTR-pShuttle (Clontech) the open reading framehaving the 3′-UTR was prepared by amplification from the DNA ofadenovirus type 5 (E1B55k forward primer=5′-ATGGAGCGAAGAAACCC-3′ andE1B55k 3′UTR backward primer=5′-CACGTCCTGGAAAAAATACAC-3′) and introducedin the blunt ended NheI restriction site, which was provided with T-ends(TA-cloning) and cloned into the pShuttle plasmid of the companyClontech. Thus, the transgene was provided with a hCMV-promoter at the5′end and with the bovine growth hormone polyadenylation signal at the3′end.

Cloning of the Vector E4ORF6-IRES-pcDNA3.1(+)

The amplificates E4orf6 using the adenovirus type 5 DNA as template(E4orf6 forward primer 5′-CTTCAGGATCCATGACTACGTCCGGCG-3′ and E4orf6backward primer 5′-GAAGTGAATTCCTACATGGGGGTAGAGTCATAATCGT-3′) and fromthe plasmid pCMVE4-34 kD which has been cut with Bam HI (Dobbelstein etal., EMBO, 16, 4276-4284, 1997), and the IRES element having thepCITE-4a(+) of the company Novagen as template (IRES forwardprimer=5′-TCCGGTTATTTTCCACCATATTGC-3′ and IRES backwardprimer=5′-TTATCATCGTGTTTTTCAAAGG-3′) were subsequently cloned into themultiple cloning site of the pcDNA3.1(+)-vector. For such purpose,primers were used for the E4orf6 transgene which create a BamHI cleavagesite at the 5′-end and a EcoRI cleavage site at the 3′-end of the openreading frame. The amplificate was digested with the respectiverestriction enzymes and the ends thereof were made compatible for thedirected cloning into the vector which has been opened using BamHI andEcoRI. Subsequently, plasmid E4orf6 in pcDNA3.1(+) was linearized withEcoRV, the T-ends added and the amplificate cloned into the IRESelement. After checking the correct orientation of the IRES element, thevector was used for further cloning.

The linkage of both transgenes with the IRES element resulted from acloning of the E4orf6-IRES cassette into the previously generatedplasmid CMV-E1B55k 3′UTR-polyA-pShuttle (Clontech) which was linearizedwith NotI, blunt ended and subsequently cut with XbaI. E4orf6-IRES inpcDNA3.1 (+) was linearized with NotI, the ends made blunt ended andfurther digested with NheI. By ligating the E4orf6-IRES insert with theCMV-E1B55k 3′UTR-polyA-pShuttle (Clontech) XVIR-3′UTR was generated inpShuttle (Clontech).

Generation of the Used Adenoviral Shuttle Vector

As the shuttle vector pΔE1sp1A, now used for the adenoviral generationsystem of the company Microbix, did neither contain a CMV promoter nor abovine growth hormone polyadenylation signal, these elements were clonedinto pΔE1sp1A. For such purpose, pΔE1sp1A was linearized with ClaI, madeblunt ended and cut with EcoRI. The element CMV-MCS (multiple cloningsite)-poly-A was linearized from pShuttle (Clontech) with MfeI, the endsmade blunt ended and further cut with EcoRI. Subsequently, the cassette(Xvir-3′UTR pShuttle from Clontech) was cloned with PmeI into theCMV-MCS-poly-A pΔE1sp1A vector which had also been cut with PmeI andsubsequently dephosphorylated. The cloning product Xvir-3′UTR-pΔE1sp1Awas used for virus generation.

Virus Generation

Xvir-3′UTR-pΔE1sp1A and pBHGE3 (from Microbix, contains the E3-regionwhich corresponds to wildtype adenovirus type 5) was cotransfected intoHEK 293 cells, whereupon virus Ad-Xvir-3′UTR E3 was generated due torecombination of homologous sequences of both vectors.

Generation of Adenovirus Ad-Xvir3′UTR-AdEASY E3 using the AdEASY-System(Company Qbiogene)

Generation of the Used Adenoviral Shuttle Vector

As, for the present used system, the vector pShuttle-AdEASY did neithercontain a CMV-promotor nor the bovine growth hormone polyadenylationsignal, these elements were cloned into pShuttle-AdEASY. For suchpurpose, the plasmid was digested with EcoRI, the ends made blunt endedby fling them up with T4-polymerase and dNTPs, the backbone wasdephosphorylated and both of the generated digestion products ligatedagain. By doing so the restriction recognition site for EcoRI waseliminated. The thus resulting plasmid was referred to aspShuttle(-EcoRI)-AdEASY.

Subsequently, the cassette CMV-MCS-polyA from the pShuttle of Clontechwas cut wich MfeI and EcoRI, the ends made blunt ended and cloned intothe vector pShuttle (-EcoRI)-AdEASY which was, for such purpose,linearized with XbaI, made blunt ended and dephosphorylated. Thusplasmid CMV-MCS-polyA-pShuttle-AdEASY was generated. The cassetteE4Orf6-IRES-E1B55k-3′UTR was cloned into this plasmid using MluI andEcoRI. By doing so the plasmid Xvir-3′UTR in pShuttle AdEASY wasgenerated. This was linearized with Bstl 1071 and MroI and introducedinto BJ5183 (EC) bacteria together with rescue-plasmid pAdEASY by meansof electroporation. By homologous recombination the adenoviral plasmidAd-Xvir-3′UTR-pAdEASY was generated which resulted in virus productionafter transfection in HEK293 cells.

Introducing the wt E3 Region into pAdEASY

As the E3 region is substantially deleted in plasmid pAdEASY, the E3region was cloned from plasmid pAdEASY with SpeI and PacI into plasmidCMV-MCS-polyA pShuttle (AdEASY) for reconstruction and thus the plasmidE3E4-pShuttle-AdEASY generated.

By restriction with NdeI and religation one out of two NdeI restrictionsites was deleted and so was the multiple cloning site from the plasmid.By this procedure plasmid E3E4-pShuttle(-NdeI)-AdEASY was generated.

Subsequently the 4007 bp wtE3-region fragment from wildtype adenovirustype 5 was excised by SpeI and NdeI and cloned into the E3E4-pShuttle(-NdeI)-AdEASY which was opened by SpeI and NdeI. The thus generatedvector was referred to as wtE3E4-pShuttle (NdeI)-AdEASY.

Subsequently the wildtype E3E4-region from the E3E4-pShuttle(-NdeI)-AdEASY was cut with SpeI and PacI and cloned into the pAdEASYand cut with SpeI and PacI, whereby in plasmid pAdEASY the E3-region wasre-established (pAdEASY-E3). XVir-3′UTR-pAdEASY-E3 was generated byhomologous recombination upon transforming BJ5183 (EC) bacteria withplasmids Xvir-3′UTR in pShuttle AdEASY and pAdEASY-E3.

Manipulation of E4 for all of the Systems Mentioned

In order to provide space for therapeutic genes and transgenes and inorder to avoid undesired homologous recombination the E4 region inplasmid E3E4-pShuttle (-NdeI)-AdEASY can be deleted specifically. Forsuch purpose, the E4orf6 region is shortened by about 0.6 kB, preferably634 bp, by excision with PstI and religation. This can, as described inFIG. 17, be performed in connection with Xvir03/01. Respective deletionsare also feasible by the one skilled in the art in different systems forthe generation of recombinant adenovirus.

Cloning of the RGD-motif in Ad-Xvir 3′UTR-AdEASY E3 in Particular (AlsoApplicable to Other Systems)

For increasing the infectivity the HI Loop of the fibre knob domain wasmodified following Dmitriev et al. 1998 (An Adenovirus Vector withGenetically Modified Fibers Demonstrates Expanded Tropism viaUtilization of a Coxsackievirus and Adenovirus Receptor-Independent CellEntry Mechanism): The respective region was amplified using the primersRGD-Hpa fw (5′-GAGgttaacCTAAGCACTGCCAAG-3′), RGD-EcoRV rev(5′-CATAGAGTATGCAGATATCGTTAGTGTTACAGGTTTAGTTTTG-3′) and RGD-EcoRV fw(5′-GTAACACTAACGATATCTGCATACTCTATGTCATTTTCATGG-3′) and RGD-Bfr rev(5′-CAGCGACATGAActtaagTGAGCTGC-3′) and thus an EcoRV restriction sitegenerated. In this restriction site the paired oligonucleotides werecloned which code for an Arg-Gly-Asp (RGD)-peptide: RGD-oligo 1(5′-CACACTAAACGGTACACAGGAAACAGGAGACACAACTTGTGACTGCCGCGGAGACTGTTTCTGCCC-3′) and RGD-oligo 2 (5′-GGGCAGAAACAG TCTCCGCGGCAGTCACAAGTTGTGTCTCCTGTTTCCTGTGTACCGTTTAGTGTG-3′). Thus, the RGD motif ispresent in the HI Loop of the fibre knob domain.

The vector described above is in principle suitable as are the otherviruses described herein for use in accordance with the presentinvention. In particular the afore-described vector is suitable toreplicate and trigger lysis insofar, in cells which are YB-1nucleus-positive cells as well as in cells where YB-1 is deregulated,i.e. is overexpressed compared to normal cells and non-tumor cells,respectively. The use of this vector particularly applies to thosediseases and groups of patients or groups of patients which aredisclosed in connection with the other adenoviruses which are describedherein to be used in accordance with the present invention and the otheradenoviruses of the present invention disclosed herein.

EXAMPLE 12 Structural Design of the Adenoviral Vector Xvir03/01

As may be taken from FIG. 17, Xvir03/01 is a further development ofXvir03. Therapeutic genes such as, for example, the genes describedherein and the transgene can be cloned into the E3 region. Additionally,a deletion was introduced into the E4 region so as to avoid homologousrecombination with the E4orf6 from the expression cassette of Xvir03.This allows that larger transgenes can be cloned in this construct. Thedeleted E3 region contains SacI, NdeI and NheI cleavage sites forintroducing a cassette, into which, for example, the therapeutictransgenes can be cloned. However, the E3 region may also stay intactand the therapeutic genes may be clines into the E4 region. Thus, amongothers, the expression of the adenoviral death protein ADP is ensured.

Preparing a Plasmid for Cloning Therapeutic Genes into the E3 Region asWell as for Making Deletions in the E4 Region: System Adeno-X ofClontech

The pAdenoX-Plasmid of Clontech has a restriction site for SfuI behindthe 3′ ITR region which is absent in wildtype adenovirus. The E3-E4region was taken from pAdenoX (Clontech) with the SpeI (position 23644)and SfuI and transferred into pcDNA3.1(+)(Invitrogen)=pcDNA3.1-E3Δ27865-30995-E4. The bigger part of E4ORF6,namely 33241-33875 was removed by means of PstI=pcDNA3.1-E3Δ27865-30995,E4Δ33241-33875. For the further development of Xvir03 the deleted E3/E4region from pcDNA3.1-E3Δ27865-30995, E4Δ33241-33875 was cloned by meansof SfuI and SpeI into plasmid pAdenoX pAdenoX E3Δ27865-30995,E4Δ33241-33875.

The expression cassette was subsequently, as described for Xvir03,cloned with I-Ceu I and PI-SceI from the E1B55k-IRES-E4orf6-pShuttletogether with the CMV promoter and the bovine growth hormone (BGH)-PolyAinto pAdenoX E3Δ27865-30995, E4Δ3241-33875 and referred to asAdcmvE1B/IRES/E4orf6-ΔE4. Subsequently, the adenovirus was prepared inaccordance with manufacturer's instructions (Clontech).

It is within the present invention and feasible for he one skilled inthe art in the light of the present disclosure that other systems nay beused for the manufacture of the adenoviruses in accordance with thepresent invention and in particular the recombinant adenoviruses, suchas the systems of the companies QBIOGENE and Nicrobix.

The afore-described vector is in principle useful as are the otherviruses described herein to be used in accordance with the presentinvention. In particular the afore-described vector is suitable toreplicate in YB-1 nucleus-positive cells as well as cells in which YB-1is deregulated, i.e. is overexpressed compared to normal cells andnon-tumor cells, and to cause lysis insofar. This vector can also beused for those diseases and groups of patients and collectives ofpatients which are disclosed herein for the other adenoviruses to beused in accordance with the present invention and the adenoviruses inaccordance with the present invention.

EXAMPLE 13 Oncolytic Effect of Xvir 03 in 257 RDB and 181 RDB Cells

100,000 cells (257RDB and 181RDB) were plated per well of a plate havingsix wells (engl.: 6 well plate). AT the next day the cells were, asdepicted in FIG. 18, infected with Ad312 (20 pfu/cell) and Xvir03 (5pfu/cell). The infection was performed in 500 μl serum free DMEM mediumat 37° C. for 1 h. Subsequently, the infection medium was removed andreplaced by 2 ml complete medium (10% FCS/DMEM). The analysis was doneby means of crystal violet staining after 5 days. The result is depictedin FIGS. 18A and 18B.

As may be taken from FIGS. 18A and 18B, the multidrug resistant cellswhich have YB-1 in the nucleus, show lysis after infection with Ad312and Xvir03 only in case of Xvir03 as represented by the crystal violetstaining of the cells. In connection therewith, first the medium isremoved. Subsequently the cells are covered with crystal violet (50%ETOH, 3% formaldehyde, 5% acetic acid, 1% crystal violet) and incubatedat room temperature for 5-10 min. Subsequently, the six well plates arethoroughly rinsed with water and dried at room temperature.

It is known to the present inventor that E1A-deleted viruses (e.g.Ad312) which, however, are not transactivating adenoviruses in the senseof the present invention, may replicate very efficiently at higher MOIs(Nevins J. R., Cell 26, 213-220, 1981), which, however, cannot berealised in clinical application. This phenomenon is referred to in theliterature as “E1A-like activity”. The adenovirus Ad312 as used herein,is an E1A-deleted virus. At the titer used (20 pfu/cell), which is stillabove the clinically desirable titer, the early adenoviral genes such asE1B55k and E4orf6 are not expressed or expressed only to a very smallextent (Nevins J. R., Cell 26, 213-220, 1981). As already describedherein, these genes and proteins play an important role in viralreplication. In contrast thereto, these genes and proteins,respectively, are expressed by adenovirus Xvir03 (FIG. 16). As may betaken from FIGS. 18A and 18B, the expression of the genes E1B55k andE4orf6 will result in an efficient viral replication and cell lysis at aconcomitantly lower infection titer required (expressed as pfu/cell).This confirms the finding underlying the present invention, namely thatthe expression of E4orf6 and E1B-55K (and the absence of E1A) incombination with nuclear localisation of YB-1 is capable of inducing avery efficient adenoviral replication. The titer required therefor ofonly 1 to 5 pfu/cell now allows for clinical application.

This confirms the finding underlying the present invention, namely thatthe presence of YB-1 in the nucleus, particularly the presenceindependent from the cell cycle, is required in order to make theviruses which are to be used in accordance with the present invention,lyse infected cells.

EXAMPLE 14 Northern Blot Analysis of the E2 Gene Expression ofAdenovirus Ad312

In each case 1 million A549 and U2OS cells were plated in 10 cm Petridishes. At the next day the cells were infected with Ad312 (50 pfu/cell)and Adwt (which served as control, 5 pfu/cell). The high virus titer ofAd312 which was used resulted in an E1-independent replication in tumorcells. The infection was done in 1-2 ml serum-free DMEM medium for 1 hat 37° C. Subsequently, the infection medium was removed and replaced by10 ml complete medium (10% FCS/DMEM). After 3 days the RNA was isolated.Subsequently, the concentration of the isolated RNA was measured in aphotometer at 260 nm. Then the RNA samples were electrophoreticallyseparated in a 0.8% formaldehyde agarose gel. Subsequently, the RNA wasblotted on a nylon membrane (conducted according to the system ofSchleicher & Schuell). The RNA blotted on the membrane is blottedagainst an “early probe” E2 and a “late probe” E2. The 1501 bp “lateprobe” specifically binds behind the E2-late promoter. The probe wasprepared prior to that by PCR (primer: 5′-GTC GGA GAT CAG ATC CGC GT(SEQ. ID. NO. 4), 5′-GAT CCT CGT CGT CTT CGC TT (SEQ. ID. NO. 5)) andradioactively labelled using ³²P. In contrast, the early probe bindsbetween the E2-early promoter and the E2-late promoter (position:226791-227002) and was also generated by means of PCR (primer:5′-AGCTGATCTTCGCTTTTG (SEQ. ID. NO. 6), 5′-GGATAGCAAGACTCTGAC AAAG (SEQ.ID. NO. 7)). Subsequently, the membrane was washed and exposed to afilm.

The result is depicted in FIG. 19. Both the early as well as the lateprobe provided specific signals in the control infection with wildtypeadenovirus, whereas tumor cells infected with Ad312 only provided aspecific signal when the late probe was used. This confirms the findingunderlying the present invention that the expression of E4orf6 andE1B55K and the absence of E1A transports overexpressed and deregulatedYB-1, respectively, into the nucleus and thus induces E2 gene expressionas a prerequisite for efficient adenoviral replication.

EXAMPLE 15 Northern Blot Analysis of the E2 Gene Expression ofAdenovirus Addelta 24

In each 1 million U2OS cells were plated in 10 cm Petri dishes. At thenext day the cells were infected with adenovirus delta 24 (Addelta24)(10 pfu/cell) and wildtype adenovirus (Adwt) (served as a control, 10pfu/cell). The used recombinant adenovirus Addelta24 (Fueyo, J. et al.,Oncogene 19, 2-12, 2000) has a specific deletion in the CR2 region ofthe E1A protein and is thus only capable of replicating in Rb-negativetumors. Additionally, the virus expresses the genes E1B55k and E4orf6comparable to the wildtype adenovirus. The infection occurred in 1-2 mlserum-free DMEM medium for 1 h at 37° C. Subsequently, the infectionmedium was removed and replaced by 10 ml complete medium (10% FCS/DMEM).The RNA was isolated after 12 h and 24 h. Subsequently, theconcentration of the isolated RNA was determined in a photometer at 260nm. Then the RNA samples were electrophoretically separated in a 0.8%formaldehyde agarose gel. Subsequently, the RNA was blotted on a nylonmembrane (conducted according to the system of Schleicher & Schuell).The RNA blotted onto the membrane is hybridised against the “earlyprobe” and against the “late probe”. The “late probe” comprising 1501bp, binds specifically behind the E2-late promoter. The probe wasprepared prior to that by PCR (primer: 5′-GTC GGA GAT CAG ATC CGC GT(SEQ. ID. NO. 4), 5′-GAT CCT CGT CGT CTT CGC TT (SEQ. ID. NO. 5)) andradioactively labelled using ³²P. The early probe, however, bindsbetween the E2-early promoter and the E2-late promoter and was alsoprepared by PCR (primer: 5′-AGCTGATCTTCGCTTTG (SEQ. ID. NO. 6),5′-GGATAGCAAGACTCTGACAAAG (SEQ. ID. NO. 7)). Subsequently, the membranewas washed and exposed to a film.

The result is shown in FIG. 20.

After 12 h only the late probe provided for a specific signal. Onlyafter 24 h also the early probe provided a signal in cells infected withAddelta24. Compared to wildtype adenoviruses, however, the signal issignificantly weaker. Also this result confirms the finding underlyingthe present invention that the expression of E4orf6 and E1B-55Ktransports overexpressed and deregulated YB-1, respectively, into thenucleus which subsequently binds to the E2-late promoter and induces E2gene expression.

EXAMPLE 16 Structural Design of the Adenoviral Vectors XvirPSJL1 andXvirPSJL2

Description of the vectors: The vectors of the XvirPSJL group which areembodiments of the viruses referred to herein as group I adenovirusesand which are exemplified by the vectors and adenoviruses, respectively,XvirPSJL1 and XvirPSJL2, are not only, like adenovirus dl520, capable ofreplicating in YB-1 nucleus-positive cells, in particular tumor cells,but also in tumor cells in which YB-1 is overexpressed and deregulated,respectively. While the viral genes E1B55k and E4orf6 are expressed onlyin dl520 infected YB-1 nucleus-positive cells under the influence of theE1B promoter and the E4 promoter, respectively, the expression of E1B55kand E4orf6 in XvirPSJL occurs by means of the cytomegalovirus (cmw)promoter. Instead of the cmw promoter, however, also other promoters, inparticular tumor-specific, tissue-specific and organ-specific promotersand the natural E1A promoter, i.e. preferably the E1A promoter aspresent in wildtype adenovirus, preferably Ad5, may be used. Because ofthe expression of E1B55k and E4orf6 the overexpressed YB-1 and thederegulated YB-1, respectively, is transported into the nucleus andadenoviral replication is initiated. The adenoviral vectors of theXvirPSJL group as disclosed herein, thus combine various elements andthus functions of the adenoviral vectors dl520, Xvir03 and AdYB-1 in asingle vector. Similar to the vector dl520 the XvirPSJL viruses containthe E1A12S gene. This gene and the corresponding gene product,respectively, is responsible for the induction of the S phase of theinfected cell and promotes viral replication and the effect ofchemotherapeutics and irradiation. Like Xvir03 the XvirPSJL virusescontain the expression cassette CMV-E4orf6/IRES/E1B55k, which isrequired for an efficient replication and indirectly or directlytransports deregulated YB-1 into the nucleus which is preferablycontained in tumor cells. Thus replication is possible only in cells,particularly tumor cells, where YB-1 is overexpressed or deregulated.Additionally, P53 is made subject to degradation by the E1B55k/E4orf6complex. The sequence coding for human transcription factor YB-1 istaken from the virus AdYB-1. The endogenous, i.e. the YB-1 alreadypresent in the cell amplifies viral replication. The expression of bothE1A12S and YB-1 is controlled by the YB-1-dependent adenoviral E2-latepromoter. Also in connection therewith specific promoters may be used,in particular tumor-specific, tissue-specific or organ-specificpromoters. A further feature of these viruses is that the E4 region isdeleted. The vector contains restriction sites there by which, in caseof the adenoviral vectors XvirPSJL1 and XvirPSJL2, various transgenes asdisclosed in the specification such as ribozymes, antisense molecules,siRNA, apoptosis-inducing genes, cytokines and prodrug genes may beexpressed. Their expression may also be controlled by tumor-specific,tissue-specific or organ-specific promoters as disclosed in thespecification. The localisation of the expression cassettes is notfixed, particularly not with regard to or within the E1, E3 and E4region, but can be arranged in any way. In connection therewith thenon-required can be either deleted or can be intact. The vectorsreplicate independent of the p53 or Rb status of the tumor cells.

The structural designs of the recombinant adenoviruses XvirPSJL1 andXvirPSJL2 are presented in FIGS. 21 and 22:

Generation of the vector XvirPSJL according to the system of Aden-X ofClontech.

Generation of the Cassette E2-late-YB11RES/12S:

The pAdenoX plasmid of Clontech/BD Biosciences which is used as astarting material herein, comprises the genomic nucleic acid ofadenovirus Ad5 and has a SfuI restriction site behind the 3′ ITR regionwhich is ABSENT in wildtype adenovirus. The E3-E4 region was transferredby SpeI (position 23644) and SfuI from pAdenoX (Clontech) intopcDNA3.1(+) (Invitrogen) and referred to as pcDNA3.1-E3Δ27865-30995-E4.The majority of the E4ORF6, namely the bases 33241-33875 were removed bymeans of PstI. The such obtained fragment was referred to aspcDNA3.1-E3Δ27865-30995, E4Δ33241-33875.

The E2-late promoter was excised from pGL3-EGFP (Holm et al., JBC 2002,277, 10427-10434) with SacI and NheI and cloned intopcDNA3.1-E3Δ27865-30995, E4Δ33241-33875. In doing so, the E3 region wasfurther deleted in the region of bases A27593-31509. The thus obtainedfragment was referred to as E2-late-pcDNA3.1-E3Δ27593-31509,E4Δ33241-33875

The cDNA for the E1A-243AA product was generated by means of RT-PCR,isolated and the sequence checked and cloned into the pcDNA3.1(+) vector(Invitrogen) using BamHI and EcoRI. E1A-12S-pcDNA3.1+was linearised withNheI and BamHI, made blunt-ended by T4 polymerase and provided with Toverhangs by Taq polymerase and dTTPs. The IRES element was cloned as aPCR product (template=pCITE, Novagen) into the E1A-12S-pcDNA 3.1(+)vector (TA cloning strategy).

The YB-1-EcoRI fragment was isolated from the vector pHVad2c (Holm etal., JBC 2002, 277, 10427-10434) and made blunt-ended. The vectorpShuttle (commercially available from BD Biosciences) was linearisedwith XbaI, the ends made blunt-ended and dephosphorylated and ligatedwith the previously produced YB-1 coding nucleic acid. The vector thusobtained was referred to as YB-1-pShuttle. The cloning into the pShuttlevector provided the YB-1 fragment coding nucleic acid with an in-frameSTOP codon. The YB-1 coding nucleic acid was cloned from theYB-1-pShuttle by means of NheI and BfrI into the vector IRES-E1A-12S inpcDNA3.1 (+). The thus obtained fragment was referred to as YB-1(EcoRI-EcoRI with STOP codon)-IRES-E1A-12S-pcDNA3.1 (+).

Subsequently, the cassette YB-1-IRES-E1A12S was excised with PmeI andcloned into the NheI linearised, blunt-ended and dephosphorylated vectorE2late-pcDNA3.1 E3Δ27593-31509, E4Δ33241-33875. Thus the second cassetteis in the deleted region of the E3 region.

The transgene cassette comprising the nucleic acid constructE2late-YB-1-IRES-E1A12S was cloned together with the remainingadenoviral sequences E3Δ27593-31509, E4Δ33241-33875 by means of SfuI andSpeI into the vector pAdenoX of Clontech(=AdenoX/E2late-YB-1-IRES-E1A12S/E3Δ27593-31509, E4Δ33241-33875).

The cassette CMV-E1B55k/IRES/E4orf6 was excised by means of 1-CeuI andPI-SceI from the pShuttle described above in relation to Xvir03 andinserted into the vector AdenoX/E2late-YB-1-IRES-E1A12S/E3Δ27593-31509,E4Δ33241-33875.

Subsequently, the vector was linearised with Pac I, transfected into 293cells and the recombinant adenovirus XvirPSJL 1 and XvirPSJL 2,respectively, isolated without the transgenes indicated in the figure inaccordance with manufacturer's instructions.

It is within the present invention and feasible for the one skilled inthe art in the light of the present disclosure that other systems may beused, such as the system of the companies QBIOGENE and MICROBIX, for thegeneration of the adenoviruses in accordance with the present invention,preferably recombinant adenovirus and in particular those containing,separately and/or together, the cassettes E4orf6-IRES-E1B55k andE1A12S-IRES-YB-1, respectively. Additionally, the individual transgenescan be exchanged within the individual cassettes and in particular amongthe respective cassettes. Additionally, the cassette E1A12S-IRES-YB-1may consist only of EA12S and/or E1A12S can be linked to other relevantgenes through IRES.

Generation of the Adenovirus AdPSJL-E2-Late Promoter 12S-AdEASY withE1A12S in the Deleted E3-Region with the AdEASY-System (CompanyMicrobix).

Cloning of PSJL 12S

First, the E2-late promoter was cloned into the HindIII and BglIIcleavage site of the pGL3-enhancer plasmid (pGL3-E2-late) as pairedoligonucleotides (upper primer5′-TCGAGCTCCGCATTTGGCGGGCGGGATTGGTCTTCGTAGAACCTAATCTCGTGGGCGTGGTAGTCCTCAGGTACAAAT-3′ and lower primer5′-AGCTTATTTGTACCTGAGGACTACCACGCCCACGAGATTAGGTTCTACGAAGACCAATCCCGCCCGCCAAATGCGGAGC-3′).

Subsequently, the luciferase gene was excised using NcoI and XbaI, theends made blunt ended and T-ends added. The transgene E1A 12S which wasamplified by the primers E1A 12S forward primer 5′-ATGGCCGCCAGTCTTTTG-3′and E1A 12S backward primer 5′-TTATGGCCTGGGGCGTTTAC-3′, was introducedby TA-cloning into the thus opened site.

This cassette was excised using PvuI and ClaI, the ends made blunt endedand cloned into the blunt ended and dephosphorylated NheI-cleavage sitein the E3-region of E3E4-pShuttle (-NdeI)-AdEASY. The cassette thuscontains the E2-late promoter, the open reading frame E1a-12S and theSV-40 late polyadenylation signal. The resulting construct isE2-late-E1a-12S-E3E4-pShuttle(-NdeI)-AdEASY.

Subsequently the E2-late-E1a 12S-E3E4 was excised from the E2-late-E1a12S-E3E4-pShuttle (-NdeI)-AdEASY using SpeI and PacI and cloned into theSpeI and PacI cut pAdEASY. The thus resulting construct was referred toas E2-late-E1a 12S-E3E4-pAdEASY. AdPSJL-12S-AdEASY was generated byhomologous recombination upon transforming BJ5183 (EC) bacteria with theplasmids Xvir-3′UTR in pShuttle AdEASY and E2-late-E1a 12S-E3E4-pAdEASY.

Generation of the Adenovirus AdPSJL-E2-Late Promoter-12S-YB-1-AdEASYwith E1A12S and YB-1 in the Deleted E3-Region Using the AdEASY System(Company Microbix)

Cloning of the vector E4ORF6-IRES-pcDNA3.1(+)

The amplificates E1a 12S (see above) and the IRES element (see above)were subsequently cloned into the multiple cloning site of thepcDNA3.1(+)-vector. For such purpose the E1a-12S amplificate wasintroduced into the blunt ended BamHI-cleavage site by TA-cloning.Subsequently, the plasmid E1a-12S in pcDNA3.1(+) was linearized withEcoRV, T-ends added and the amplificate cloned into the IRES element.The thus obtained plasmid was subsequently linearized with XhoI, theends made blunt ended and the EcoRI-EcoRI-cleavage product of YB-1 whichis devoid of a stop codon.

The thus created construct E1A-12S-IRES-pcDNA3.1(+) was linearized usingNotI and the ends made blunt ended. Also, the YB-1-EcoRI-cleavageproduct was made blunt ended and introduced into the dephosphorylatedvector E1A-12S-IRES-pcDNA3.1(+). The cassette E1A-12S-IRES-YB-1 wasremoved using PmeI and cloned into the above described plasmidpGL3-E2-late after removal of the liciferase gene with NcoI and XbaI andblunt ending and dephosphorylation.

The cassette E2-late-E1A-12S-IRES-YB-1 was excised using PvuI and ClaI,the ends made blunt ended and cloned into the blunt ended anddephosphorylated NheI-cleavage site in the E3-region of E3E4-pShuttle(-NdeI)-AdEASY. The thus obtained construct is E2-latepromoter-E1A-12S-IRES-YB-1-E3E4-pShuttle (-NdeI)-AdEASY.

Subsequently, the E2-late promoter-E1A-12S-IRES-YB-1-E3E4 cassette wasexcised from the E2-late promoter-E1A-12S-IRES-YB-1-E3E4-pShuttle(-NdeI)-AdEASY with SpeI and PacI and cloned into the SpeI and PacI cutpAdEASY. The resulting construct was referred to asE1a-12S-IRES-YB-1-E3E4-pAdEASY.

AdPSJL-12S-Yb-1-AdEASY was generated by homologous recombination upontransformation of BJ5183 (EC) bacteria with the plasmid Xvir-3′UTR inpShuttle AdEASY and E1a-12S-IRES-YB-1-E3E4-pAdEASY.

Cloning of the Cassette E2-Late Promoter-E1A-12S and/or E2-LatePromoter-E1A-12S-IRES-YB-1 in the E4-Region

After manipulation and deletion, respectively, of the E4 region usingPstI 634 bp were removed. The cassettes E2-late promoter-E1A-12S and/orE2-late promoter-E1A-12S-IRES-YB-1 can be introduced into the E4-region.Alternatively, the E2-region may remain intact under such conditions.

Cloning of the RGD-Motive

For an improved infectivity the HI loop of the fibre knob domain wasmodified according to Dmitriev et al. 1998 (An Adenovirus Vector withGenetically Modified Fibers Demonstrates Expanded Tropism viaUtilization of a Coxsackievirus and Adenovirus Receptor-Independent CellEntry Mechanism): The respective region was amplified using the primesRGD-Hpa fw (5′-GAGgttaacCTAAGCACTGCCAAG-3′), RGD-EcoRV rev(5′-CATAGAGTATGCAGATATCGTTAGTGTTACAGGTTTAGTTTTG-3′), as well asRGD-EcoRV fw (5′-GTAACACTAACGATATCTGCATACTCTATGTCATTTTCATGG-3′) andRGD-Bfr rev (5′-CAGCGACATGAActtaagTGAGCTGC-3′) and an EcoRV-cleavagesite thus generated. Paired oligonucleotides were cloned into thiscleavage site which code for an Arg-Gly-Asp (RGD)-peptide with RGD oligo1 (5′-CACACTAAACGGTACACAGGAAACAGGAGACACAACTTGTGAC TGCCGCGGAGACTGTTTCTGCCC-3′) and RGD oligo 2 (5′-GGGCAGAAACAGTCTCCGCGGCAGTCACAAGTTGTGTCTCCTGTTTCCTGTGTACCGTTTAGTGTG-3′). Thus the RGDmotif is contained in the HI loop of the fibre knob domain.

In FIGS. 30 and 31 the cloned E1B55k-3′UTR is described in more detailand the pE3/E4 Shuttle plasmid in the Adeasy system is depicted in FIG.32. The plasmid is characterised in that manipulations and deletions,respectively, have been made to regions E3 and E4 which allow to clonedifferent cassettes by means of different restriction site such as NheIfor the E3 region and PstI for the E4 region without adversely affectingthe opent reading frames other than the one of E4orf6 and L5.Additinoally, a sequence for the RGD motif is introdiced into the regionof the HI loop.

Positions of the deletions corresponding to the wildtype adenovirussequence.

E3 deletion: 28138-30818

E4 deletion: 33246-33875

RGD motif: 32678 (9 amino acids introduced, central 3 amino acids RGD:CDCRGDCFC

EXAMPLE 17 Infection of HeLa Cells with Adenovirus dl520

100.000 HeLa cells were plated per dish. At the next day the cells wereinfected with various titers (pfu/ml) of adenovirus dl520. The infectionwas done in 500 μl serum-free DMEM medium for 1 h at 37° C.Subsequently, the infection medium was removed and replaced by 2 mlcomplete medium (10% FCS/DMEM). After 3-5 days an analysis was performedusing crystal violet staining.

The result of this experiment is depicted in FIG. 23. The adenovirusdl520 does not show any lysis at low MOIs (5-10 pfu/cell) upon infectionof HeLa cells which do not have YB-1 in the nucleus. In contrastthereto, dl520 showed a factually complete lysis at an MOI (multiplicityof infection) of 100-200 pfu per cell and a still predominant lysis atan MOI of 50 pfu per cell. Therefrom it can be concluded that dl520 andsimilar viruses which are capable of switching on the adenoviral genesE1B55k and E4orf6 at higher MOIs, are suitable to transport eitherdirectly or indirectly overexpressed or deregulated YB-1 into thenucleus and thus to induce cell lysis.

EXAMPLE 18 Luciferase Assay for Determining the E2-Late PromoterActivity

It is known that YB-1 binds to the adenoviral E2-late promoter in thenucleus (Holm et al., JBC 2002, 277, 10427-20434) and that this promoteris also well suited for the expression of nucleic acids. The use of theadenoviral E2-late promoter is particularly motivated by the fact thatit can be regulated by YB-1, whereby YB-1 acts as a positive effector,i.e. the promoter is only active in the presence of YB-1 in the nucleus.Insofar said adenoviral E2-late promoter can be regulated in a highlyselective manner and thus used in systems in which YB-1 is present inthe nucleus and factually avoids any expression of the nucleic acidwhich is under the control of the adenoviral E2-late promoter in casethat YB-1 is not present in the nucleus as an effector and regulator,respectively. The E2-late promoter comprises 3 Y-boxes (CCAAT) which arerelevant for the activation of the E2 gene. Different E2-late promoterconstructions have been prepared and tested for their specificity andactivity. The analysis was carried out as follows.

The cell lines EPG-257 RDB (epithelial stomach carcinoma) which has YB-1in the nucleus, HeLa (epithelial uterine cervix carcinoma) and U2OS(osteosarcoma) were seeded using three different cell concentrations in6 well plates. The wells which showed confluence of 70% at the next day,were used for transfection. For each well 500 ng SpinMiniprep (Qiagen)purified plasmid DNA of the different E2-late promoter constructions inluciferase vectors (commercially available from Promega, startingplasmid: pGL3-enhancer) were added to 500 μl OptiMEM in a 1.5 ml lockingcap reaction vessel and 5 μl DOTAP to 500 μl in a further locking capreaction vessel. Both solutions were combined and mixed. The mixture wasincubated for complex formation for 30 minutes at room temperature. Thecells were rinsed three times with PBS and covered with a layer of thetransfection mixture. The plates were incubated at 37° C. for 5 hours,subsequently rinsed again three times with PBS and provided withcomplete medium.

The cells were processed with the Luciferase Assay System Kit of Promega(Cat. No. E11500) 48 h after infection: Each well was provided with alayer of 500 μl lysis buffer, the cells rinsed off from the well platewith a 1 ml pipette after 10 minutes at room temperature and transferredinto a 1,5 ml locking cap reaction vessel. The cell lysate wassubsequently centrifuged at 4° C. for 15 minutes at 14.000 rpm. To each50 μl of the supernatant 100 μl luciferase substrate were added andmeasured with TopCount (Canberra-Packard GmbH, 63303 Dreieich)Microplate Scintillation & Luminescence counter in black plates with 96wells at a wave length of 945 nm.

Protein was measured with the BCA Protein Assay Reagent Kit, cataloguenumber 23227 (PIERCE, Rockford, Ill., USA) at 570 nm in a bioluminometer(Biolumin™ 960) kinetic fluorescence/absorbance plate reader ofMolecular Dynamics. The relative light signals of the samples weretranslated into the protein amount (RLU/μg protein).

The following plasmids were used: pGL3-enhancer (Promega) from which theenhancer was removed by means of BamHI (2250 bp) and BsaBI (2003 bp),served as a blank reading. The various E2 promoter constructions werecloned into the MCS in the enhancer-lacking pGL3 vector by means ofrestriction sites Apa I and Sac I. The hCMV promoter was cloned by meansof Bgl II and Hind III into the pGL3 enhancer and served as a positivecontrol. The positive control allowed to estimate transfectionefficiency and also served as a reference value for luciferase activity.For each cell line the CMV control was set 100% and the enzyme activityproduced by the E2 promoter constructions put in relation thereto anddepicted as a bar graph in FIG. 24.

The various constructs were referred to as follows:

1. comprising the Y-box I, II and III corresponding to bases 25932-26179bp (referring to the wildtype adenovirus sequence, see also the part ofthe subsequently provided adenoviral E2 region)

2. comprising the Y-box II and III corresponding to bases 25932-26127 bp(referring to the wildtype adenovirus sequence, see also the part of thesubsequently provided adenoviral E2 region)

3. comprising the Y-box III corresponding to bases 25932-26004 bp(referring to the wildtype adenovirus sequence, see also the part of thesubsequently provided adenoviral E2 region)

4. comprising no Y-box as acting as the blank reading

Part of the Adenoviral E2 Region (Taken from Virology 1992, 186,280-285)

(The YB-1 binding sites are printed in bold): 25561aggaactttatcctagagcgctcaggaatcttgcccgccacctgctgtgcacttcctagc 25621gactttgtgcccattaagtaccgcgaatgccctccgccgctttggggccactgctacctt 25681ctgcagctagccaactaccttgcctaccactctgacataatggaagacgtgagcggtgac 25741ggtctactggagtgtcactgtcgctgcaacctatgcaccccgcaccgctccctggtttgc 25801aattcgcagctgcttaacgaaagtcaaattatcggtacctttgagctgcagggtccctcg 25861cctgacgaaaagtccgcggctccggggttgaaactcactccggggctgtggacgtcggct 25921

25981

26041

26101

26161 tatcagcagcagccgcgggcccttgcttcccaggatggcacccaaaaagaagctgcagct 26221gccgccgccacccacggacgaggaggaatactgggacagtcaggcagaggaggttttgga 26281cgaggaggaggaggacatgatggaagactgggagagcctagacgaggaagcttccgaggt 26341cgaagaggtgtcagacgaaacaccgtcaccctcggtcgcattcccctcgccggcgcccca 26401gaaatcggcaaccggttccagcatggctacaacctccgctcctcaggcgccgccggcact 26461gcccgttcgccgacccaaccgtagatgggacaccactggaaccagggccggtaagtccaa 26521gcagccgccgccgttagcccaagagcaacaacagcgccaaggctaccgctcatggcgcgg 26581gcacaagaacgccatagttgcttgcttgcaagactgtgggggcaacatctccttcgcccg 26641ccgctttcttctctaccatcacggcgtggccttcccccgtaacatcctgcattactaccg 26701tcatctctacagcccatactgcaccggcggcagcggcagcggcagcaacagcagcggcca 26761cacagaagcaaaggcgaccggatagcaagactctgacaaagcccaagaaatccacagcgg (SEQ. ID.No. 8)

The results presented in FIG. 24 confirm in an impressive manner thatthe individual promoter fragments which contain differentE2-late/Y-boxes, are suitable for the expression of therapeutictransgenes in YB-1 nucleus-positive tumor cells and may thus be used aspromoters in the meaning of the present invention.

EXAMPLE 19 Effect of Yb-1 Expressed by Adenovirus on Particle Release

Human osteosarcoma cells (U2OS) were infected with the E1/E3-deletedadenoviral vector AdYB-1 and Ad312 only having E1A-deleted, at an MOI of50 pfu/cell. AdYB-1 contains in its genome the sequence coding for thecellular transcription factor YB-1 and thus expresses the Y-box bindingprotein 1 (YB-1). In order to evaluate the release of viral particles as“plaque forming units” (pfu) after infection, either the supernatant ofthe culture medium or the remaining cell layer was isolated 2 and 5days, respectively, post infectionem. The intracellular particles werereleased by 3 cycles of thawing/freezing. The particle number wasanalysed using the plaque assay on 293 cells.

The result is in depicted in FIG. 25, whereby the solid bars indicatethe intracellular remaining viral particles, whereas the cross-stripedbars represent the released, extracellular viral particles.

The result depicted in FIG. 25 confirms that AdYB-1, as a whole,produces more pfu than Ad312 and releases more particles. After 5 daysthe AdYB-1 infected cells clearly show a cytopathic effect (CPE) incontrast to Ad312-infected cells.

EXAMPLE 14 Replication of Adenovirus in Cells after Addition ofIrinotecan

In order to determine the effect of Irinotecan on adenoviral replication10⁶ U373 tumour cells were plated in 10 cm² Petri dishes. In a firstreaction 5 μM Irinotecan was added after 24 hours. After another 24hours the cells were infected with 10 pfu/cell dl520. After incubationof 3 days without Irinotecan DNA was isolated in accordance with theprocedure described in example 10.

In a parallel reaction the thus prepared U373-cells were notpre-incubated with Irinotecan. After 48 hours of cultivating the cellswithout Irinotecan, they were infected with 10 pfu/cell dl520 andsubsequently incubated without Irinotecan for another 3 days. DNA wasisolated as described above.

Subsequently 2 μg DNA were digested with restriction enzyme Kpn I and aSouthern Blot analysis performed. A part of the adenoviral genome(position:22734-24235) generated by means of PCR was used as a probe.

The result is depicted in FIG. 26. FIG. 26 shows that after incubationwith Irinotecan adenoviral replication is significantly increased inU373 cells after treatment with Irinotecan (lane 2) compared tountreated control where no incubation with Irinotecan was performed(lane 1). This means that adenoviral replication is increased under theinfluence of Irinotecan.

EXAMPLE 15 Replication of Adenovirus in Cells after Administration ofTrichostatin a

In order to test the effect of Trichostatin A on adenoviral replication,10⁶ U373 tumour cells were plated in 10 cm² Petri dishes. After 24 hours0.25, 0.5 and 0.75 μM Trichostatin A was added. After another 24 hoursthe cells were infected with 10 pfu/cell dl520.

After 3 days of incubation in medium without Trichostatin DNA wasisolated. Subsequently 2 μg DNA were digested with restriction enzymeKpn I and a Southern Blot analysis performed. A part of the adenoviralgenome (position:22734-24235) generated by means of PCR was used as aprobe.

The result is depicted in FIG. 27. FIG. 27 shows that after incubationwith increasing concentrations of Trichostatin A adenoviral replicationin U373 cells (lanes 2, 3 and 4) is significantly increased compared tountreated controls where no incubation with Trichostatin A was performed(lane 1). This means that viral replication is increased under theinfluence of Trichostatin A.

EXAMPLE 16 Influencing the Expression ofCoxsackievirus-Adenovirus-Receptor (CAR) on U373 Cells in Response toAddition of Trichostatin A

200,000 U373 cells were plated in 6 well plates. After 24 hours thecells were cultivated with 1 μM Trichostatin for 24 hours. After another24 hours the cells were isolated. Subsequently, analysis of CARexpression was performed according to a standard protocol usingFacs-analysis and the primary antibody anti-CAR clone RmcB from thecompany Upstate, and a rabbit-anti-mouse FITC as secondary antibody(company DAKO).

The result is depicted in FIG. 28. Without Trichostatin treatment 11.3%of the cells were CAR-positive, whereby after incubation of the cellswith 1 μM Trichostatin 56.2% of the cells were CAR-positive. The figuresare percentages of the overall cells used in the test.

From FIG. 28 it can be taken that under the influence of the histonedeacylase inhibitor Trichostatin A CAR, which is an important factor forthe binding of adenovirus, is expressed at a higher level and moreavailable, respectively, which increases the efficacy of transfection ofthe thus treated cells.

EXAMPLE 23 Oncolysis of U373 Cells by Adenovirus after CombinedTreatment of the Cells with Irinotecan and Trichostatin A

200,000 U373 cells were plated in a 6 well plate. After 24 hours either2 μM Irinotecan or only 1 μM Trichostatin A or 1 μM Irinotecan +0.5 μMTrichostatin were added to the medium. After 24 hours of incubation thecells were infected with 10, 20 and 30 pfu/cell dl520. After 3-5 daysthe analysis was performed using crystal violet staining. The assayswere performed in duplicate.

The result is depicted in FIG. 29. The six plates represented in panel 1show a complete cell layer which was not affected by incubation with acombination of Irinotecan and Trichostatin A as shown by crystal violetstaining. The next two wells of panel 1 show the cell layer afterinfection with 10 and 20 pfu/cell dl520, respectively. Also under suchconditions there is no lysis of the cells which is due to the absence ofreplication of dl520. Thus it is shown that neither dl520 at 10 or 20pfu/cells nor 1 μM Irinotecan +0.5 μM Trichostatin A alone are suitableto induce cell lysis.

The further 6 well plates 2, 3 and 4 depicted in FIG. 29, herein alsoreferred to as panels 2, 3 and 4, were basically treated in accordancewith this scheme. The individual wells were inoculated with U373 cellsas previously described and the cells cultivated therein. The wells wereinoculated with 10, 20 or 30 pfu/cell dl520 in duplicate, whereby thedifference between the three 6 well plates resided in the kind ofcytostatics used. In panel 2 2 μM Irinotecan, in panel 3 1 μMTrichostatin A and in panel 4 1 μM Irinotecan and 0.5 μM Trichostatin Awas added to the individual wells.

In the 6 well plate 2 (panel 2) with 2 μM Irinotecan the cells werelysed with 30 pfu/cell dl520. In the 6 well plate 3 (panel 3) with 1 μMTrichostatin A the cells were lysed at 20 and 30 pfu/cell dl520. In the6 well plate 4 (panel 4) with 1 μM Irenotecan +0.5 μM Trichostatin A thecells, in contrast thereto, were already lysed at 10 pfu/cell dl 520.

The test, the results of which are depicted in FIGS. 26 to 29, showsthat the combination consisting of Irinotecan+Trichostatin A+dl520induces a more effective cell lyses of tumour cells as any compoundalone. This results, on the one hand, from Trichostatin A increasingCAR-expression and thus significantly improves infectability of thecells. On the other hand, Irinotecan translates YB-1 into the cellnucleus and thus induces an improved adenoviral replication.Additionally, the cellular YB-1 is assisting adenoviral replicationafter infection with dl520 and is no longer available for DNA-repairprocesses. Depending on the point of view, this results in an improvedefficacy of dl520 on the one hand and an increased efficacy of thecytostatics on the other hand.

The features of the invention disclosed in the preceding specification,the claims as well as the figures can both individually as well as inany combination be important to the realisation of the invention in itsvarious embodiments.

1. Adenovirus expressing a first protein which is selected from thegroup comprising an E1B protein and an E4 protein, prior to a secondprotein which is selected from the group comprising an E1A-protein. 2.Adenovirus according to claim 1 characterised in that the first proteinis an E1B protein, preferably an E1B55 kd protein.
 3. Adenovirusaccording to claim 1, characterised in that the first protein is an E4protein, preferably an E4orf6 protein.
 4. Adenovirus according to any ofclaims 1 to 3, characterised in that the first protein is a combinationof E1B protein and E4 protein, preferably a combination of E1B55 kDprotein and E4orf6 protein.
 5. Adenovirus according to any of claims 1to 4, characterised in that the E1A protein is an E1A12S protein. 6.Adenovirus, preferably an adenovirus according to any of claims 1 to 5,characterised in that the adenovirus comprises at least one nucleic acidcoding for a protein which is selected from the group comprising E1Bproteins, E4 proteins and E1A proteins, whereby the at least one proteinis under the control of a promoter which is different from the promotercontrolling the expression of the protein in a wildtype adenovirus. 7.Adenovirus according to claim 6, characterised in that the at least oneprotein is an E1B protein, preferably an E1B55 kD protein.
 8. Adenovirusaccording to claim 6 or 7, characterised in that the at least oneprotein is an E4 protein, preferably an E4orf6 protein.
 9. Adenovirusaccording to any of claims 6 to 8, characterised in that the at leastone protein is an E1A protein, preferably an E1A12S protein. 10.Adenovirus according to any of claims 6 to 9, characterised in that theat least one protein is a combination of E1B protein and E4 protein,preferably a combination of E1B55kD protein and E4orf6 protein. 11.Adenovirus according to any of claims 6 to 9, characterised in that theat least one protein is a combination of E1B protein and E1A protein,preferably a combination of E1B55 kD protein and E1A12S protein. 12.Adenovirus according to any of claims 6 to 9, characterised in that theat least one protein is a combination of E4 protein and E1A protein,preferably a combination of E4orf6 protein and E1A12S protein. 13.Adenovirus according to any of claims 6 to 9, characterised in that theat least one protein is a combination of E1B protein, E4 protein and E1Aprotein, preferably a combination of E1B55 kD protein, E4orf6 proteinand E1A12S protein.
 14. Adenovirus according to any of claims 6 to 13,characterised in that the expression of the E1B protein is controlled bya promoter, whereby the promoter is selected from the group comprisingtumor-specific promoters, organ-specific promoters, tissue-specificpromoters, heterologous promoters and adenoviral promoters, whereby theadenoviral promoter is different from the E1B promoter.
 15. Adenovirusaccording to any of claims 6 to 14, characterised in that the expressionof the E4 protein is controlled by a promoter, whereby the promoter isselected from the group comprising tumor-specific promoters,organ-specific promoters, tissue-specific promoters, heterologouspromoters and adenoviral promoters, whereby the adenoviral promoter isdifferent from the E4 promoter.
 16. Adenovirus according to claims 14 or15, whereby the adenoviral promoter is the E1A promoter.
 17. Adenovirusaccording to any of claims 6 to 16, characterised in that the expressionof the E1A protein is controlled by a promoter, whereby the promoter isselected from the group comprising tumor-specific promoters,organ-specific promoters, tissue-specific promoters, heterologouspromoters and adenoviral promoters, whereby the adenoviral promoter isdifferent from the E1A promoter.
 18. Adenovirus according to any ofclaims 14 to 17, characterised in that the promoter controlling theexpression of the E1A protein is YB-1 controlled or can be regulated byYB-1.
 19. Adenovirus according to any of claims 14 to 18, characterisedin that the promoter controlling the expression of the E1A protein isthe adenoviral E2 late promoter.
 20. Adenovirus according to any ofclaims 1 to 19, characterised in that the E4 protein, preferably theE4orf6 protein, and the E1B protein, preferably the E1B55 kd protein,are under the control of the same or a common promoter.
 21. Adenovirus,preferably an adenovirus according to any of claims 1 to 20,characterised in that the adenovirus provides YB-1 in the nucleusthrough at least one adenoviral protein or that the provision of YB-1 inthe nucleus is mediated through at least one adenoviral protein, wherebypreferably the adenoviral protein is different from E1A.
 22. Adenovirus,preferably according to any of claims 1 to 21, characterised in that theadenovirus provides YB-1 for adenoviral replication through at least oneadenoviral protein or mediates the provision of YB-1 for adenoviralreplication through at least one adenoviral protein, whereby preferablythe adenoviral protein is different from E1A.
 23. Adenovirus accordingto claim 21 or 22, characterised in that the adenoviral protein is acomplex of E4orf6 and E1B55 kd.
 24. Adenovirus, preferably according toany of claims 1 to 23, characterised in that the nucleic acid of theadenovirus comprises at least one functionally inactive adenoviralregion, whereby the region is selected from the group comprising the E1region, the E3 region, the E4 region and combinations thereof. 25.Adenovirus according to claim 24, characterised in that the region isthe E1 region.
 26. Adenovirus according to claim 24 or 25, characterisedin that the region is the E3 region.
 27. Adenovirus according to any ofclaims 24 to 26, characterised in that the region is the E4 region. 28.Adenovirus according to any of claims 24 to 27, characterised in thatthe region comprises the E1 region, the E3 region and the E4 region. 29.Adenovirus, preferably an adenovirus according to any of claims 1 to 28,characterised in that the adenovirus comprises at least one expressioncassette, whereby the expression cassette comprises at least onepromoter and a nucleic acid coding for an adenoviral protein, wherebythe adenoviral protein is an E1B protein, preferably an E1B55 kDprotein.
 30. Adenovirus according to claim 29, characterised in that thepromoter is different from the E1B promoter.
 31. Adenovirus according toclaim 30, characterised in that the promoter is selected from the groupcomprising tumor-specific promoters, organ-specific promoters,tissue-specific promoters, heterologous promoters and adenoviralpromoters, whereby the promoter is different from the E1B promoter. 32.Adenovirus, preferably according to any of claims 1 to 31, characterisedin that the adenovirus comprises at least one expression cassette,whereby the expression cassette comprises at least one promoter and anucleic acid coding for an adenoviral protein, whereby the adenoviralprotein is an E4 protein, preferably an E4orf6 protein.
 33. Adenovirusaccording to claim 32, characterised in that the promoter is differentfrom the E4 promoter.
 34. Adenovirus according to claim 33,characterised in that the promoter is selected from the group comprisingtumor-specific promoters, organ-specific promoters, tissue-specificpromoters, heterologous promoters and adenoviral promoters, whereby theadenoviral promoters are different from the E4 promoter.
 35. Adenovirusaccording to any of claims 29 to 34, characterised in that the promoteris the E1A promoter.
 36. Adenovirus, preferably according to any ofclaims 1 to 35, characterised in that the adenovirus comprises at leastone expression cassette, whereby the expression cassette comprises atleast one promoter and a nucleic acid coding for an adenoviral protein,whereby the adenoviral protein is an E1A protein, preferably an E1A12Sprotein.
 37. Adenovirus according to claim 36, characterised in that thepromoter is different from the E1A promoter.
 38. Adenovirus according toclaim 37, characterised in that the promoter is selected from the groupcomprising tumor-specific promoters, organ-specific promoters,tissue-specific promoters, heterologous promoters and adenoviralpromoters.
 39. Adenovirus according to any of claims 1 to 38,characterised in that the adenovirus comprises a nucleic acid, wherebythe nucleic acid codes for YB-1.
 40. Adenovirus according to claim 39,characterised in that the nucleic acid coding for YB-1 is under thecontrol of a promoter, whereby the promoter is preferably the E2 latepromoter.
 41. Adenovirus according to claim 39 or 40, characterised inthat the nucleic acid coding for YB-1 is under the control of apromoter, whereby the promoter is YB-1 dependent and YB-1 controlled,respectively.
 42. Adenovirus according to any of claims 35 to 41,characterised in that the nucleic acid coding for YB-1 is part of theexpression cassette comprising a nucleic acid coding for an E1A protein,preferably a nucleic acid coding for an E1A12S protein.
 43. Adenovirusaccording to claim 42, characterised in that the nucleic acid coding forthe E1A protein is separated from the nucleic acid coding for YB-1through an IRES sequence.
 44. Adenovirus according to any of claims 29to 43, characterised in that the nucleic acid coding for the E4 protein,preferably the E4orf6 protein, and the nucleic acid coding for the E1Bprotein, preferably the E1B55 kD protein, are contained in an expressioncassette, whereby preferably the two coding sequences are separatedthrough an IRES sequence.
 45. Adenovirus according to claim 44,characterised in that the promoter of the expression cassette isselected from the group comprising tumor-specific promoters,organ-specific promoters, tissue-specific promoters, heterologouspromoters and adenoviral promoters, whereby the adenoviral promoters aredifferent from the E4 promoter and different from the E1B promoter,preferably different from the wildtype E4 promoter and different fromthe wildtype E1B promoter.
 46. Adenovirus according to any of claims 1to 45, characterised in that the adenovirus comprises an expressioncassette comprising a promoter and a nucleic acid sequence, whereby thenucleic acid sequence is selected from the group comprising aptamers,ribozymes, aptazymes, antisense molecules and siRNA.
 47. Adenovirusaccording to any of claims 1 to 45, characterised in that the adenoviruscomprises an expression cassette comprising a promoter and a nucleicacid sequence, whereby the nucleic acid sequence is a coding nucleicacid, whereby the nucleic acid codes for a molecule which is selectedfrom the group comprising peptides, polypeptides, proteins, anticalines,antibodies and antibody fragments.
 48. Adenovirus according to any ofclaims 1 to 45, characterised in that the adenovirus comprises anexpression cassette, whereby the expression cassette comprises apromoter and a nucleic acid sequence, whereby the nucleic acid sequenceis selected from the group comprising apoptosis inducing genes, prodruggenes, protease inhibitors, tumor suppressor genes, cytokines andangiogenesis inhibitors.
 49. Adenovirus according to any of claims 1 to48, characterised in that the adenovirus is a recombinant adenovirus.50. Adenovirus according to any of claims 1 to 49, characterised in thatthe adenovirus is an adenovirus mutant.
 51. Adenovirus according to anyof claims 1 to 50, characterised in that the adenovirus is replicationdeficient.
 52. Adenovirus according to claim 51, characterised in thatthe adenovirus is capable of replicating in cells comprising deregulatedYB-1 or having YB-1 in the nucleus.
 53. Adenovirus according to claim52, characterised in that the cells contain YB-1 in the nucleusindependent of the cell cycle.
 54. Nucleic acid coding for an adenovirusaccording to any of claims 1 to
 53. 55. Replication system comprising anucleic acid according to claim 54 and a nucleic acid of a helper virus,whereby the nucleic acid of the helper virus comprises one or more ofthe expression cassettes of the adenovirus according to any of claims 1to
 53. 56. Replication system according to claim 55, characterised inthat the adenovirus or the nucleic acid coding therefor is lacking theexpression cassette comprised by the helper virus.
 57. Vector comprisinga nucleic acid according to claim 54 and/or a replication systemaccording to any of claims 55 to
 56. 58. Vector according to claim 57,characterised in that the vector is an expression vector.
 59. Cellcomprising an adenovirus according to any of claims 1 to 53 and/or anucleic acid according to claim 54 and/or a replication system accordingto claim 55 or 56 and/or a vector according to claim 57 or
 58. 60. Cellaccording to claim 59, characterised in that the cell is a eucaryoticcell, preferably an animal cell, more preferably a mammalian cell. 61.Cell according to claim 60, characterised in that the mammalian cell isa cell selected from the group comprising cells of mice, rats, guineapigs, pigs, sheep, goats, cattle, horses, dogs, cats and human beings.62. Organism, preferably a mammal organism, comprising an adenovirusaccording to any of claims 1 to 53, a nucleic acid according to claim54, a replication system according to claim 55 or 56, a vector accordingto any of claims 57 or 58 or a cell according to any of claims 59 to 61,whereby the organism is preferably selected from the group comprisingmice, rats, guinea pigs, pigs, sheep, goats, cattle, horses, dogs andcats.
 63. Use of an adenovirus according to any of claims 1 to 53, anucleic acid according to claim 54, a replication system according toclaim 55 or 56, a vector according to any of claims 57 or 58, or a cellaccording to any of claims 59 to 61, for replication of an adenovirus,preferably for in vitro replication of an adenovirus.
 64. Use of anadenovirus according to any of claims 1 to 53, a nucleic acid accordingto claim 54, a replication system according to claim 55 or 56, a vectoraccording to any of claims 57 or 58, or a cell according to any ofclaims 59 to 61 for the manufacture of an adenovirus, preferably for invitro manufacture of an adenovirus.
 65. Use of an adenovirus accordingto any of claims 1 to 53, a nucleic acid according to claim 54, areplication system according to claim 55 or 56, a vector according toany of claims 57 or 58, or a cell according to any of claims 59 to 61for the expression of genes, preferably of genes which promote celllysis, preferably cell lysis during adenoviral replication, and/or arepromoting adenoviral mediated cell lysis.
 66. Use of an adenovirusaccording to any of claims 1 to 53, a nucleic acid according to claim54, a replication system according to claim 55 or 56, a vector accordingto any of claims 57 or 58, or a cell according to any of claims 59 to 61for the manufacture of a medicament.
 67. Use according to any of claims63 to 66, characterised in that the cell in which the adenovirusreplicates, has YB-1 in its nucleus, preferably has YB-1 in its nucleusindependent of the cell cycle.
 68. Use according to any of claims 63 to66, characterised in that the cell in which the adenovirus replicates,comprises deregulated YB-1.
 69. Use according to claim 66, characterisedin that the medicament is for the treatment of tumor diseases.
 70. Useaccording to claim 69, characterised in that the tumor disease isselected from the group comprising malignant diseases, cancer, cancerdiseases and tumors.
 71. Use according to claim 70, characterised inthat the tumors are selected from the group comprising solid, non-solid,malignant and benign tumors.
 72. Use according to any of claims 69 to71, characterised in that at least one part of the tumor forming cellshave YB-1 in the nucleus, preferably have YB-1 in the nucleusindependent of the cell cycle.
 73. Use according to any of claims 69 to72, characterised in that at least a part of the cells forming the tumorcomprises deregulated YB-1.
 74. Use according to any of claims 69 to 73,characterised in that at least a part of the cells forming the tumor areRb positive or Rb negative.
 75. Use according to any of claims 69 to 73,characterised in that at least a part of the cells forming the tumorhave a resistance, preferably a multiple resistance againstpharmaceutically active agents.
 76. Use according to claim 75,characterised in that the resistance is a multiple resistance.
 77. Useaccording to any of claims 75 or 76, characterised in that theresistance is against anti-tumor agents, preferably cytostatics, and/orthat the resistance is caused by irradiation.
 77. Use according to anyof claims 69 to 76, characterised in that the patient for which themedicament is intended, comprises a plurality of cells, whereby thecells are cells as described in any of claims 72 to
 76. 78. Useaccording to any of claims 69 to 77, characterised in that themedicament comprises at least one further pharmaceutically active agent.79. Use according to any of claims 68 to 77, characterised in that themedicament is administered together with a further pharmaceuticallyactive agent or is intended therefor.
 80. Use according to claim 78 or79, characterised in that the further pharmaceutically active agent isselected from the group comprising cytokines, metalloproteinaseinhibitors, angiogenesis inhibitors, cytostatics, tyrosine kinaseinhibitors, cell cycle inhibitors, proteosome inhibitors, inhibitors ofthe signal transduction cascade, protein kinases and recombinantantibodies.
 81. Use according to any of claims 69 to 77, characterisedin that the medicament is administered prior, during or afterirradiation.
 82. Use according to claim 81, characterised in that theradiation is administered for the purpose of treating a tumor.
 83. Useaccording to any of claims 69 to 82, characterised in that the cell orthe organism to be treated is subject to a measure, whereby the measureis selected from the group comprising irradiation, administration ofcytostatics and hyperthermia.
 84. Use according to any of claims 69 to83, characterised in that the measure is applied locally orsystemically.
 85. Use according to any of the preceding claims,characterised in that the irradiation uses high-energy radiation,preferably uses any irradiation as used in the treatment of tumordiseases.
 86. Use of an adenovirus according to any of claims 1 to 53, anucleic acid according to claim 54, a replication system according toclaim 55 or 56, a vector according to any of claims 57 or 58, or a cellaccording to any of claims 59 to 61 for the manufacture of a medicamentfor the treatment of tumor diseases, characterised in that the tumordisease is selected from the group comprising breast tumors, bonetumors, gastric tumors, intestinal tumors, gall-bladder tumors, pancreastumors, liver tumors, kidney tumors, brain tumors, ovarian tumors, skintumors, tumors of cutaneous appendages, head and neck cancer, uterinetumors, synovial tumors, laryngeal tumors, oesophageal tumors, lingualtumors, prostate tumors, preferably one of the preceding tumor diseaseshaving the characteristics as described in any of the preceding claims.87. Use of an adenovirus according to any of claims 1 to 53, a nucleicacid according to claim 54, a replication system according to claim 55or 56, a vector according to any of claims 57 or 58, or a cell accordingto any of claims 59 to 61 for the manufacture of medicament for thetreatment of tumor diseases, whereby the tumor-specific promoter is apromoter which is specific for the tumor for which the medicament isused.
 88. Pharmaceutical composition comprising an adenovirus accordingto any of claims 1 to 53, a nucleic acid according to claim 54, areplication system according to claim 55 or 56, a vector according toany of claims 57 or 58, or a cell according to any of claims 59 to 61and optionally a pharmaceutically acceptable carrier.
 89. Use accordingto any of the preceding claims, characterised in that the medicamentcomprises a combination of at least two agents, whereby each agent isindividually and independently selected from the group comprisingcytostatics.
 90. Use according to claim 89, characterized in that atleast two of the agetns address different target molecules.
 91. Useaccording to claim 90, characterized in that at least two of the agentsare active by a different mode of action.
 92. Use according to any ofclaims 89 to 91, characterized in that at least one agent increases theabilitiy of a cell to be infected, whereby the virus replicates in suchcell.
 93. Use according to any of claims 89 to 92, characterized in thatat least one agent influences the availability of a component of thecell, preferably increases the availability of the component, wherebythe component mediates the uptake of the virus.
 94. Use according to anyof claims 89 to 93, characterized in that at least one agent mediatesthe transport of YB-1 into the nucleus, preferably increases saidtransport.
 95. Use according to any of claims 89 to 94, characterized inthat at least one agent is a histone deacylase inhibitor.
 96. Useaccording to claim 95, characterized in that the histone deacylaseinhibitor is selected from the group comprising Trichostatin A, FR901228, MS-27-275, NVP-LAQ824, PXD101 Apicidin and Scriptaid.
 97. Useaccording to any of claims 89 to 95, characterized in that at least oneagent is selected from the group comprising Trichostatin A, FR 901228,MS-27-275, NVP-LAQ824, PXD101, Apicidin and Scriptaid.
 98. Use accordingto any of claims 89 to 97, characterized in that at least one agent is atopoisomerase inhibitor.
 99. Use according to claims 98, characterizedin that the topoisomerase inhibitor is selected from the groupcomprising Camptothecin, Irinotecan, Topotecan, DX-8951f, SN-38,9-aminocamptothecin, 9-nitrocamptothecin, Daunorubicn and Etoposid. 100.Use according to any of the proceeding claims, characterized in that theagent comprises Trichostatin A and Irinotecan.
 101. Use according to anyof the proceeding claims, characterized in that the virus, in particularthe virus according to any of the proceeding claims, is separated fromthe at least two agents.
 102. Use according to claim 68, characterizedin that at least one unit dosis of the virus is separated from at leastone unit dosis of one or the at least two agents.
 103. Kit comprising avirus, preferably a virus according to any of the proceeding claims, andat least two agents, whereby any agent is individually and independentlyselected from the group comprising cytostatics.